Substituted amino acids as erythropoietin mimetics

ABSTRACT

This invention relates to a series of substitituted amino acids of Formula I  
                 
 
     pharmaceutical compositions containing them and intermediates used in their manufacture. The compounds of the invention are small molecules which bind to the erythropoietin receptor and compete with the natural ligand for binding to this receptor.

[0001] This invention relates to a series of small molecules which bindto the erythropoietin receptor and compete with the natural ligand forbinding to said receptor. The invention includes pharmaceuticalcompositions containing these mimetics, their methods of production aswell as intermediates used in their synthesis.

[0002] Erythropoietin (EPO) is a 34,000 dalton glycoprotein hormonewhich is produced in the mammalian kidney. Its primary role isstimulation of mitotic cell division and differentiation of erythrocyteprecursor cells. As a result this hormone regulates the production oferythrocytes, the hemoglobin contained therein and the blood's abilityto carry oxygen. The commercial product Epogen® is used in the treatmentof anemia. This drug is produced by recombinant techniques and isformulated in aqueous isotonic sodium chloride/sodium citrate. Eventhough it has been used successfully in the treatment of anemia, it is acostly drug that is administered intravenously. This method ofadministration is both costly and inconvenient for the patient;therefore it would be desirable to find a EPO mimetic which has thepotential for oral activity.

[0003] A small molecule EPO mimetic has advantages over the naturalprotein. The immune response associated with large peptides is unlikelyto occur with small molecules. In addition, the variety ofpharmaceutical formulations that may be used with small molecules aretechnically unfeasible for proteins. Thus the use of relatively inertformulations for small molecules is possible. The most importantadvantage of small molecules is their potential for oral activity. Suchan agent would ease administration, cost less and facilitate patientcompliance.

[0004] Although compounds which mimic EPO are useful in stimulating redblood cell synthesis, there are diseases where the overproduction of redblood cells is a problem. Erythroleukemia and polysythemia vera areexamples of such diseases. Since EPO is an agent responsible for thematuration of red blood cell precursors, an antagonist of EPO would haveutility treating either of those diseases.

SUMMARY OF THE INVENTION

[0005] The disclosed invention consists of a series of small moleculeswhich demonstrate competitive binding with the natural ligand for theEPO receptor. As such these compounds are potentially useful in thetreatment of diseases or conditions associated with this receptor. Inaddition, the invention contemplates methods of producing thesecompounds and intermediates used in their production.

[0006] The invention includes compounds of the Formula I:

[0007] I

[0008] wherein:

[0009] R¹ is the side chain of a natural or unnatural ax-amino acids,where if said side chain contains a protectable group, that group may beprotected with a member of the group consisting of succinyl, glutaryl,3,3-dimethylglutaryl, C₁₋₅alkyl, C₁₋₅alkoxycarbonyl, acetyl,N-(9-fluorenylmethoxycarbonyl), trifluoroacetyl,omega-carboxyC₁₋₅alkylcarbonyl, t-butoxycarbonyl, benzyl,benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, phenylsulfonyl, ureido,t-butyl, cinnamoyl, trityl, 4-methyltrityl,1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl, tosyl,4-methoxy-2,3,6-trimethylbenzenesulfonyl, phenylureido, and substitutedphenylureido (where the phenyl substituents are phenoxy, halo,C₁₋₅alkoxycarbonyl);

[0010] R² and R³ may be taken together to form a six-membered aromaticring which is fused to the depicted ring, or are independently selectedfrom the group consisting of hydrogen, C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy,halo, trifluoromethyl, nitro, amino, phenyl, phenoxy, phenylC₁₋₅alkyl,phenyl C₁₋₅alkoxy, substituted phenyl (where the substituents areselected from C₁₋₅alkyl,

[0011] C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano, andamino),

[0012] substituted phenoxy (where the substituents are selected fromC₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano,and amino),

[0013] substituted phenylC₁₋₅alkyl (where the substituents are selectedfrom C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro,cyano, and amino),

[0014] substituted phenylC₁₋₅alkoxy (where the substituents are selectedfrom C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro,cyano, and amino), and

[0015] substituted amino (where the substituents are selected from oneor more members of the group consisting of C₁₋₅alkyl,halosubstitutedC₁₋₅alkyl, C₁₋₅alknyl, C₁₋₅alkenyl, phenyl,phenylC₁₋₅alkyl, C₁₋₅alkylcarbonyl, halo substituted C₁₋₅alkylcarbonyl,carboxyC₁₋₅alkyl, C₁₋₅alkoxyC₁₋₅alkyl, cinnamoyl, naphthylcarbonyl,furylcarbonyl, pyridylcarbonyl, C₁₋₅alkylsulfonyl, phenylcarbonyl,phenylC₁₋₅alkylcarbonyl, phenylsulfonyl, phenylC₁₋₅alkylsulfonylsubstituted phenylcarbonyl, substituted phenylC₁₋₅alkylcarbonyl,substituted phenylsulfonyl, substituted phenylC₁₋₅alkylsulfonyl,substituted phenyl, and substituted phenylC₁₋₅alkyl [where the aromaticphenyl, phenylC₁₋₅alkyl, phenylcarbonyl, phenylC₁₋₅alkylcarbonyl,phenylsulfonyl, and phenylC₁₋₅alkylsulfonyl substitutents areindependently selected from one to five members of the group consistingof C₁₋₅alkyl, C₁₋₅alkoxy, hydroxy, halogen, trifluoromethyl, nitro,cyano, and amino]);

[0016] R⁴and R⁵ may be taken together to form a six-membered aromaticring which is fused to the depicted ring, or are independently selectedfrom the group consisting of hydrogen, C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy,halo, trifluoromethyl, nitro, amino, phenyl, phenoxy, phenylC₁₋₅alkyl,phenyl C₁₋₅alkoxy, substituted phenyl (where the substituents areselected from C₁₋₅alkyl,

[0017] C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano, andamino),

[0018] substituted phenoxy (where the substituents are selected fromC₁₋₅alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano, andamino),

[0019] substituted phenylC₁₋₅alkyl (where the substituents are selectedfrom C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro,cyano, and amino),

[0020] substituted phenylC₁₋₅alkoxy (where the substituents are selectedfrom C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro,cyano, and amino), and

[0021] substituted amino (where the substituents are selected from oneor more members of the group consisting of C₁₋₅alkyl,halosubstitutedC₁₋₅alkyl, C₁₋₅alknyl, C₁₋₅alkenyl, phenyl,phenylC₁₋₅alkyl, C₁₋₅alkylcarbonyl, halo substituted C₁₋₅alkylcarbonyl,carboxyC₁₋₅alkyl, C₁₋₅alkoxyC₁₋₅alkyl, cinnamoyl, naphthylcarbonyl,furylcarbonyl, pyridylcarbonyl, C₁₋₅alkylsulfonyl, phenylcarbonyl,phenylC₁₋₅alkylcarbonyl, phenylsulfonyl, phenylC₁₋₅alkylsulfonylsubstituted phenylcarbonyl, substituted phenylC₁₋₅alkylcarbonyl,substituted phenylsulfonyl, substituted phenylC₁₋₅alkylsulfonyl,substituted phenyl, and substituted phenylC₁₋₅alkyl [where the aromaticphenyl, phenylC₁₋₅alkyl, phenylcarbonyl, phenylC₁₋₅alkylcarbonyl,phenylsulfonyl, and phenylC₁₋₅alkylsulfonyl substitutents areindependently selected from one to five members of the group consistingof C₁₋₅alkyl, C₁₋₅alkoxy, hydroxy, halogen, trifluoromethyl, nitro,cyano, and amino]);

[0022] W is selected from the group consisting of —CH═CH—, —S—, and—CH═N—;

[0023] Q is selected from the group consisting of —CH═CH—, —S—, and—CH═N—;

[0024] X is selected from the group consisting of carbonyl, C₁₋₅alkyl,C₁₋₅alkenyl, C₁₋₅alkenylcarbonyl, and (CH₂)_(m)—C(O)— where m is 2-5;

[0025] Y is selected from the group consisting of carbonyl, C₁₋₅alkyl,C₁₋₅alkenyl, C₁₋₅alkenylcarbonyl, and (CH₂)_(m)—C(O)— where m is 2-5;

[0026] n is 1, 2, or 3;

[0027] Z is selected from the group consisting of hydroxy, C₁₋₅ alkoxy,phenoxy, phenylC₁₋₅alkoxy, amino, C₁₋₅alkylamino, diC₁₋₅alkylamino,phenylamino, phenylC₁₋₅alkylamino, piperidin-1-yl

[0028] substituted piperidin-1-yl (where the substituents are selectedfrom the group consisting of C₁₋₅alkyl, C₁₋₅alkoxy, halo, aminocarbonyl,C₁₋₅alkoxycarbonyl, and oxo;

[0029] substituted phenylC₁₋₅alkylamino (where the aromaticsubstitutents are selected from the group consisting of C₁₋₅alkyl,C₁₋₅alkoxy, phenylC₁₋₅alkenyloxy, hydroxy, halogen, trifluoromethyl,nitro, cyano, and amino),

[0030] substituted phenoxy (where the aromatic substitutents areselected from the group consisting of C₁₋₅alkyl, C₁₋₅alkoxy, hydroxy,halogen, trifluoromethyl, nitro, cyano, and amino),

[0031] substituted phenylC₁₋₅alkoxy (where the aromatic substitutentsare selected from the group consisting of C₁₋₅alkyl, C₁₋₅alkoxy,hydroxy, halogen, trifluoromethyl, nitro, cyano, and amino),

[0032] —OCH₂CH₂(OCH₂ ₂CH₂)_(s)OCH₂CH₂O—,

[0033] —NHCH₂CH₂(OCH₂CH₂)_(s)OCH₂CH₂NH—,

[0034] —NH(CH₂)_(p)O(CH₂)_(q)O(CH₂)_(p)NH—,—NH(CH₂)_(q)NCH₃(CH₂)_(s)NH—,

[0035] —NH(CH₂)_(s)NH—, and (NH(CH₂)_(s))₃N, where s, p, and q areindependently selected from 1-7

[0036] with the proviso that if n is 2, Z is not hydroxy, C₁₋₅ alkoxy,amino, C₁₋₅alkylamino, diC₁₋₅alkylamino, phenylamino,phenylC₁₋₅alkylamino, or piperidin-1-yl, with the further proviso thatif n is 3, Z is (NH(CH₂)_(s))₃N. and the salts thereof.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The terms used in describing the invention are commonly used andknown to those skilled in the art. “Independently” means that when thereare more than one substituent, the substitutents may be different. Theterm “alkyl” refers to straight, cyclic and branched-chain alkyl groupsand “alkoxy” refers O-alkyl where alkyl is as defined supra. “Cbz”refers to benzyloxycarbonyl. “Boc” refers to 1-butoxycarbonyl and “Ts”refers to toluenesulfonyl. “DCC” refers to 1,3-dicyclohexylcarbodiimide,“DMAP” refers to 4-N′,N-dimethylaminopyridine and “HOBT” refers to1-hydroxybenzotriazole hydrate. “Fmoc” refers toN-(9-fluorenylmethoxycarbonyl), “DABCO” refers to1,4-Diazabicyclo[2.2.2]octane, “EDCI” refers to1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, “Dde” refers to1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl, and “TMOF” refers totrimethyl orthoformate. The side chains of α-amino acids refer to thesubstituents of the stereogenic carbon of an α-amino acid. For exampleif the amino acid is lysine, the side chain is 1-aminobutan-4-yl. Theterm natural amino acid refers to the 20 α-amino acids of the Lconfiguration which are found in natural proteins. Unnatural α-aminoacids include synthetic amino acids such as, α-aminoadipic acid,4-aminobutanoic acid, 6-aminohexanoic acid, α-aminosuberic acid,5-aminopentanoic acid, p-aminophenylalanine, α-aminopimelic acidγ-carboxyglutamic acid, p-carboxyphenylalanine, carnitine, citrulline,α,β-diaminopropionic acid, α,γ-diaminobutyric acid, homocitrulline,homoserine, and statine as well as D-configuration amino acids. The term“protectable group” refers to a hydroxy, amino, carboxy, carboxamide,guanidine, amidine or a thiol groups on an amino acid side. Compounds ofthe invention may be prepared by following general procedures known tothose skilled in the art, and those set forth herein.

[0038] The compounds of the invention may be prepared by liquid phaseorganic synthesis techniques or by using amino acids which are bound toa number of known resins. The underlying chemistry, namely, acylationand alkylation reactions, peptide protection and deprotection reactionsas well as peptide coupling reactions use similar conditions andreagents. The main distinction between the two methods is in thestarting materials. While the starting materials for the liquid phasesyntheses are the N-protected amino acids or the lower alkyl esterderivatives of either the N-protected or N-unprotected amino acids, thestarting material for the resin syntheses are N-protected amino acidswhich are bound to resins by their carboxy termini.

General Procedure for the Solid-phase Synthesis of SymmetricalNα,Nα-Disubstituted Amino Acids Scheme 1

[0039] An equivalent of an N-Fmoc-protected amino acid which is bound toa resin 1a is suspended in a suitable solvent such as DMF. This solventis removed and the nitrogen protecting group (Fmoc) is removed bystirring the resin bound amino acid with an organic base, such aspiperidine, and an addition portion of the solvent. A solution of abouttwo to three equivalents of an appropriately substituted halide, 1b, anda suitable base such DIEA is added to the resin bound amino acid andthis mixture is shaken for 18-36 h. The resulting mixture is washed withseveral portions of a suitable solvent and is suspended and shaken in anacidic solution, such as 50% TFA/CH₂CH₂, over several hours to cleavethe acid from the resin and give the N-disubstituted amino acid 1c.

[0040] By varying the resin bound amino acid 1a, one may obtain many ofthe compounds of the invention. The following resin bound amino acidsmay be used in Scheme I: alanine,N-g-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)arginine,β-(4-methyltrityl)asparagine, aspartic acid (β-t-butyl ester),S-(trityl)cysteine, γ-(4-methyltrityl)glutamine, glutamic acid(β-t-butyl ester), glycine, N-imidazolyl-(trityl)histidine, isoleucine,leucine, N-ε-(2-chlorobenzyloxycarbonyl)lysine,N-ε-(t-butoxycarbonyl)lysine, methionine, phenylalanine, proline,O-(t-butyl)serine, O-(t-butyl)threonine,N-indolyl-(t-butoxycarbonyl)tryptophan, O-(t-butyl)tyrosine, valine,β-alanine, α-aminoadipic acid, 4-aminobutanoic acid,, 6-aminohexanoicacid, α-aminosuberic acid, 5-aminopentanoic acid, p-aminophenylalanine,α-aminopimelic acid γ-carboxyglutamic acid, p-carboxyphenylalanine,carnitine, citrulline, α,β-diaminopropionic acid, α,γ-diaminobutyricacid, homocitrulline, homoserine, and statine. In addition, the choiceof “W” and “X” can be varied by using known halide derivatives of 1b.For example using benzylchloride, 2-chloromethylthiophene, or2-chloromethylpyridine gives compounds of the invention where “W” is—CH═CH—, —S—, or —CH═N—, respectively. For variations in “X”, the use of2-chloroethylphenyl, 3-chloro-1-propenylbenzene, or benzeneacetylchloride as 1b, give compounds where Y is (CH₂)₂, —CH═CH—CH₂—, or—CH₂C(O)— respectively. Still further, Scheme 1 may be used to producecombinatorial mixtures of products. Using mixtures of resin bound aminoacids, 1a, with only one 1b produces said combinatorial mixtures.Alternatively, using one amino acid 1a with a mixture of 1b as well asmixture of 1a with mixtures of 1b gives a large range of combinatorialmixtures.

General Procedure for the Solid-phase Synthesis of UnsymmetricalNα,Nα-Disubstituted Amino Acids Scheme 2, Step A

[0041] An equivalent of an N-Fmoc-protected amino acid which is bound toa resin 1a is suspended in a suitable solvent such as DMF. This solventis removed and the nitrogen protecting group (Fmoc) is removed bystirring the resin bound amino acid with an organic base, such aspiperidine, and an addition portion of the solvent. Trimethylorthoformate and an appropriately substituted aldehyde 2a (5equivalents) is added and the mixture is shaken under N₂ overnight. Thismixture is treated with a suspension of NaBH(OAc)₃ (5 equivalents) inCH₂Cl₂ and shaken under N₂ overnight. After filtration and washing witha suitable solvent, the resulting product, resin boundNα-monosubstituted amino acid 2b, is rinsed with a suitable solvent andits identity is confirmed by MS and or HPLC analysis after treatmet of aportion of the resin with 50% TFA/CH₂Cl₂.

Scheme 2, Step B

[0042] The resin 2b is suspended in an appropriate solvent such as DMFand is filtered. The appropriately substituted alkyl or arylkyl halide,2c, and an appropriate base such as DIEA are added with some additionalsolvent and the mixture is shaken under N₂ for 18-36 h. The resin boundNα,Nα-disubstituted amino acid, 2d, is isolated from the suspension andthe resin is cleaved with an acidic solution to give the free acid 2e.

Scheme 3, Step C

[0043] A resin bound amine, 2d, where R⁴ is nitro, is suspended in asuitable solvent, such as DMF, and is filtered. This mixture is treatedwith SnCl₂ dihydrate in DMF and shaken under N₂ overnight. The solventis removed and the resin is washed successive portions of a suitablesolvent to give the resin bound compound 3a where R⁴ is amino. The resinis suspended in a suitable solvent and is combined with an organic base,such as pyridine an appropriately substituted carboxylic acid anhydride,acid chloride, or sulfonyl chloride. The mixture is shaken under N₂overnight and is filtered to give the resin bound amino acid 3b. Thismaterial is treated with an acid and a suitable solvent to give the freeamino acid 3b.

Scheme 3, Step D

[0044] The resin bound amine 3a is treated with TMOF and anappropriately substituted aldehyde 3c is added and the mixture is shakenunder N₂ overnight. The resulting mixture is drained and treated with asuspension of NaBH(OAc)₃ in an appropriate solvent and this mixture isshaken under N₂ overnight. The resin bound 3-aralkylaminophenyl aminoacid is identified my spectral techniques after clevage to give the freeacid 3d as previously described.

Scheme 3, Step E

[0045] Resin bound, 2d, where R¹ is (CH₂)₄NH(Dde) is mixed with asuitable solvent, such as DMF, and shaken with successive portions of 2%solution of hydrazine hydrate in DMF over about 30 min. The resin isfiltered and treated with a suitable solvent and a cyclic anhydridederivative 3e, and a base such as DMAP and pyridine. This mixture isshaken under N₂ overnight and filtered to give the resin bound amine,3f. This material is identified by spectral techniques after clevage togive the free acid 3f as previously described.

Scheme 4, Step F

[0046] Resin bound 2b, where R² is nitro is suspended in CH₂Cl₂ and istreated with an organic base, such as pyridine, and 9-fluorenylmethoxychloride. This mixture is shaken under N₂ overnight, filtered andresuspended in a suitable solvent. This mixture is treated with SnCl₂dihydrate in DMF and shaken under N₂ overnight. The solvent is removedand the resin is washed successive portions of a suitable solvent andfiltered to give the resin bound compound 4a where R² is amino. Theresin 4a is then suspended in a suitable solvent, such as CH₂Cl₂, and iscombined with 0.4 mmol of pyridine and 0.25-0.4 mmol of theappropriately substituted carboxylic acid anhydride, acid chloride, orsulfonyl chloride. The mixture is shaken under N₂ overnight, filtered,and washed successively with three portions each of CH₂Cl₂ and MeOH.This resin is suspended in DMF, filtered, and shaken under N₂ with 5 mLof a 40% solution of piperidine in DMF. After 1 h, the solvent isdrained and the resin was washed successively with three portions eachof suitable solvents to give the resin bound 4b. The identity of thecompound was confirmed by spectral analysis after cleveage as previouslydescribed.

Scheme 5

[0047] The resin 2b (0.2 mmol) is suspended in CH₂Cl₂, filtered, and isresuspended in CH₂Cl₂. This suspension is treated with diethylphosphonoacetic acid and diisopropylcarbodiimide or other suitablecarbodiimide reagent, and the mixture is shaken under N₂ overnight. Thesolvent is drained and the resulting resin 5a was washed successivelywith three portions each of CH₂Cl₂ and MeOH. The resin is suspended inDMF and filtered. A solution of the appropriately substituted aldehyde5b (0.6-1.0 mmol) in 3-5 mL of DMF, lithium bromide (0.6-1.0 mmol), anda suitable base such as DIEA or Et₃N (0.6-1.0 mmol) is added and themixture is shaken under N₂ overnight. The solvent is removed and theresin is washed successively with three portions each of DMF, CH₂Cl₂,and MeOH. The identity of the resin bound substituted amino acid 5c wasconfirmed spectral techniques. The resin bound material may be treatedwith 50% TFA/CH₂Cl₂ over 1-1.5 h, to give the acid 5c.

Scheme 6

[0048] To prepare compounds where n is 2 and Z is NH(CH₂)_(s)NH,products of Schemes 1-5 may be used in Scheme 6. Treatment of twoequivalents of the substituted amino acid 1c with an equivalent of thediamine 6a, in the presence of HOBT and a peptide coupling agent such asEDCI and a base such as DIEA at room temperature over 16 h gives thedimer 6b.

General Procedure for the Solution-phase Synthesis of SymmetricalNα,Nα-Disubstituted Amino Acids Scheme 7, Step A

[0049] A solution of of amino acid ester 7a, an appropriatelysubstituted halide derivitive 1b, and an appropriate base such as DIEA,Na₂CO₃, or Cs₂CO₃ in a suitable solvent, such as DMF, is heated at50-100° C. under N₂ overnight, or until the starting material isexhausted, to give a mixture of the di and mono-substituted amines, 7band 7c respectively. If the side chains of R¹ contain acid cleavableprotecting groups, those groups may be cleaved by treatment with 30-80%TFA/CH₂Cl₂. Esters 7b and 7c may be independently converted to thecorresponding acids 7d and 7e by hydrolysis with an appropriate basesuch as aqueous NaOH.

General Procedure for the Solution-phase Synthesis of UnsymmetricalNα,Nα-Disubstituted Amino Acids Scheme 8, Step A

[0050] A solution of 1 mmol of amino acid ester 8a (or the correspondingHCl salt and 1.1 mmol of DIEA) and 1-1.5 mmol of the appropriatelysubstituted aldehyde 2a in 3-5 mL of trimethyl orthoformate was stirredat room temperature under N₂ overnight. The solution was eitherconcentrated and used directly for the next reaction, or was partitionedbetween EtOAc and water, washed with brine, dried over Na₂SO₄, andconcentrated to give crude product, which was purified by MPLC to givemono-substituted product 8b.

Scheme 8, Step B

[0051] Amino ester 8b was dissolved in DMF, combined with 1.1-1.5 mmolof the appropriately substituted chloride or bromide 2c, and heated at50-100° C. overnight. The reaction mixture was cooled and partitionedbetween water and EtOAc. The organic layer was washed three times withwater and once with brine, dried over Na₂SO₄, and concentrated. Thecrude product was purified by MPLC to give pure 8c. For examples of 8cwherein the side chain R¹ contained an acid-cleavable protecting groupsuch as t-butylcarbamate, t-butyl ester, or t-butyl ether, 8c wasstirred in 30-80% TFA/CH₂Cl₂ for 1-3 h. The reaction mixture wasconcentrated and optionally dissolved in HOAc and freeze-dried to givethe deprotected form of 8c. For examples of 8c where R⁹ was equal tot-butyl, 8c was stirred in 30-80% TFA/CH₂Cl₂ for 1-3 h and treated asdescribed above to give acid 8d. For examples of 8c where R⁹ was equalto methyl, ethyl, or other primary or secondary alkyl esters, 8c wasstirred with with 1-2 mmol of aqueous LiOH, NaOH, or KOH in MeOH, EtOH,or THF at 20-80° C. until TLC indicated the absence of 8c. The solutionwas acidified to pH 4-5 with aqueous citric acid or HCl and wasextracted with CH₂Cl₂ or EtOAc. The organic solution was washed withbrine, dried over Na₂SO₄, and concentrated to give 8d.

Scheme 8, Step C

[0052] For examples of amino acid ester 8c where R¹=(CH₂)₄NHBoc, 8c (1mmol) was stirred in 30-80% TFA/CH₂Cl₂ for 1-3 h. The reaction mixturewas concentrated to provide 8e as the TFA salt. Optionally, the TFA saltwas dissolved in CH₂Cl₂ or EtOAc and washed with aqueous NaOH or Na₂CO₃,dried over Na₂SO₄, and concentrated to give 8e as the free base.

Scheme 8, Step D

[0053] A solution of 1 mmol of 8e, 1-4 mmol of an appropriate base suchas DIEA, and 1-2 mmol of the appropriately substituted cyclic anhydride3e was stirred in CH₂Cl₂ or DMF under N₂ overnight. The resultingmixture was diluted with CH₂Cl₂ or EtOAc and washed with aqueous HCl,water, and brine, was dried over Na₂SO₄, and concentrated to provide 8f.Alternatively, 1 mmol of 8e, 1-4 mmol of an appropriate base such asDIEA, and 1-2 mmol of the appropriately substituted carboxylic acidanhydride (R¹¹CO)₂O or acid chloride R¹¹COCl was stirred in CH₂Cl₂ orDMF under N₂ overnight and worked up as above to provide 8g.Alternatively, 1 mmol of 8e, 1-4 mmol of an appropriate base such asDIEA, and 1-2 mmol of the appropriately substituted isocyanate R¹²NCOwas stirred in CH₂Cl₂ or DMF under N₂ overnight and worked up as aboveto provide 8h.

Scheme 9, Step A

[0054] For examples of 8c where R⁵=NO₂, a solution of 1 mmol of 8c(where R², R³, R⁴, or) and 10-12 mmol of SnCl₂ dihydrate was stirred inMeOH, EtOH, or DMF at 20-80° C. for 0.5-24 h under N₂. The solution wastaken to room temperature and poured into aqueous Na₂CO₃ with rapidstirring. The resulting mixture was extracted with EtOAc or CH₂Cl₂ andthe organic extracts were washed with brine, dried over Na₂SO₄, andconcentrated to give the aminophenyl product 9a, which was purified byMPLC or used without further purification.

Scheme 9, Step B

[0055] A solution of 1 mmol of aminophenyl compound 9a and 1-1.5 mmol ofthe appropriately substituted aldehyde 2a in 3-5 mL of trimethylorthoformate was stirred at room temperature under N₂ overnight. Thesolution was either concentrated and used directly for the nextreaction, or was partitioned between EtOAc and water, washed with brine,dried over Na₂SO₄, and concentrated to give crude product, which waspurified by MPLC to give 9b. For examples of 9b wherein the side chainR¹ or R⁹ contained an acid-cleavable protecting group such ast-butylcarbamate, t-butyl ester, or t-butyl ether, 9b was stirred in30-80% TFA/CH₂Cl₂ for 1-3 h. The reaction mixture was concentrated andoptionally dissolved in HOAc and freeze-dried to give the deprotectedform of 9b.

Scheme 9, Step C

[0056] A solution of 1 mmol of 3-aminophenyl compound 9a, 1.1-2 mmol ofpyridine, and 1-1.5 mmol of the appropriately substituted acid chloride,acid anhydride, or sulfonyl chloride in 3-5 mL of CH₂Cl₂ or ClCH₂CH₂Clwas stirred at room temperature under N₂ overnight. The solution waspartitioned between EtOAc and water, washed with water, saturatedaqueous NaHCO₃, and brine, dried over Na₂SO₄, and concentrated to givecrude product which was optionally purified by MPLC to give amide orsulfonamide 9c. For examples of 9c wherein the side chain R¹ or R⁹contained an acid-cleavable protecting group such as t-butylcarbamate,t-butyl ester, or t-butyl ether, 9c was stirred in 30-80% TFA/CH₂Cl₂ for1-3 h. The reaction mixture was concentrated and optionally dissolved inHOAc and freeze-dried to give the deprotected form of 9c.

General Procedure for the Solution-phase Synthesis of SymmetricalNα,Nα-Disubstituted Amino Amides and their Dimers and Trimers Scheme 10,Step A

[0057] A solution of 1 mmol of N-Cbz-protected amino acid 10a and theappropriate amine (ZH, 1 mmol), diamine (ZH₂, 0.5 mmol), or triamine(ZH₃ 0.33 mmol), was treated with 1.1 mmol of HOBt, 1.1 mmol of DIEA,and 2.1 mmol of EDCI in 3-6 mL of CH₂Cl₂ or DMF. [Alternatively, 1 mmolof the pentafluorophenyl ester or N-hydroxysuccinimide ester of 10a wasmixed with the appropriate portion of amine (ZH), diamine (ZH₂), ortriamine (ZH₃) in 3-6 mL of DMF.] The solution was stirred at roomtemperature under N₂ for 12-24 h, and EtOAc was added. The organicsolution was washed with 5% aqueous citric acid, water, saturatedNaHCO₃, and brine, dried over Na₂SO₄, and concentrated. The crudeproduct was optionally purified by MPLC to afford amide 10b. Compound10b was stirred in 30-80% TFA/CH₂Cl₂ for 1-3 h. The reaction mixture wasconcentrated to provide the TFA salt which was dissolved in CH₂Cl₂ orEtOAc and washed with aqueous NaOH or Na₂CO₃, dried over Na₂SO₄, andconcentrated to give 10c as the free base.

Scheme 10, Step B

[0058] A solution of 1 mmol of amino acid ester 10c (n=1), 2.5-3 mmol ofthe appropriately substituted chloride or bromide 2c, and 2.5-3 mmol ofan appropriate base such as DIEA, Na₂CO₃, or Cs₂CO₃ in 3-5 mL of DMF washeated at 50-100° C. under N₂ for 18-24 h. (For examples of 10c wheren=2 or 3, the amounts of 2c and base were increased by two- orthree-fold, respectively.) The reaction mixture was cooled andpartitioned between water and EtOAc. The organic layer was washed threetimes with water and once with brine, dried over Na₂SO₄, andconcentrated. The crude product was purified by MPLC to give pure amide10d.

[0059] Alternatively, a solution of 1 mmol of amino acid ester 10c(n=1), 2.5-3 mmol of the appropriately substituted aldehyde 2a, and2.5-3 mmol of borane-pyridine complex in 3-5 mL of DMF or EtOH wasstirred at room temperature under N₂ for 3-5 days. (For examples of 10cwhere n=2 or 3, the amounts of 2c and borane-pyridine complex wereincreased by two- or three-fold, respectively.) The mixture wasconcentrated to dryness and was partitioned between water and CH₂Cl₂,washed with brine, dried over Na₂SO₄, and concentrated. The crudeproduct was purified by MPLC to give pure amide 10d.

Scheme 10, Step C

[0060] For examples of 10d where R¹=CH₂CH₂CO₂-t-Bu or CH₂CO₂-t-Bu, 10dwas stirred in 30-80% TFA/CH₂Cl₂ for 1-24 h. The reaction mixture wasconcentrated and optionally dissolved in HOAc and freeze-dried to giveacid 10e.

Scheme 10, Step D

[0061] For examples of 10d where R¹ is equal to (CH₂)₄NHBoc, 10d wasstirred in 30-80% TFA/CH₂Cl₂ for 1-24 h. The reaction mixture wasconcentrated and optionally dissolved in HOAc and freeze-dried to giveamine 10f as the TFA salt which was optionally dissolved in CH₂Cl₂ orEtOAc, washed with aqueous NaOH or Na₂CO₃, dried over Na₂SO₄, andconcentrated to give 10f as the free base.

Scheme 10, Step E

[0062] A solution of 1 mmol of 10f, 1-4 mmol of an appropriate base suchas DIEA, and 1-2 mmol of the appropriately substituted cyclic anhydride3e was stirred in CH₂Cl₂ or DMF under N₂ overnight. The resultingmixture was diluted with CH₂Cl₂ or EtOAc and washed with aqueous HCl,water, and brine, was dried over Na₂SO₄, and concentrated to provideacid 10g. Alternatively, 1 mmol of 10f, 1-4 mmol of an appropriate basesuch as DIEA, and 1-2 mmol of the appropriately substituted carboxylicacid anhydride (R¹¹CO)₂O or acid chloride R¹¹COCl was stirred in CH₂Cl₂or DMF under N₂ overnight and worked up as above to provide 10h.Alternatively, 1 mmol of 8e, 14 mmol of an appropriate base such asDIEA, and 1-2 mmol of the appropriately substituted isocyanate R¹²NCOwas stirred in CH₂Cl₂ or DMF under N₂ overnight and worked up as aboveto provide 10i.

General Procedure for the Solid-phase Synthesis of Nα,Nα-Bis-CinnamylAmino Acids and Nα-Cinnamyl Amino Acids Scheme 11

[0063] An equivalent of an N-Fmoc-protected amino acid 11a which isbound to a polystyrene resin such as Wang resin is suspended in asuitable solvent such as DMF. This solvent is removed and the nitrogenprotecting group (Fmoc) is removed by stirring the resin bound aminoacid with an organic base, such as piperidine, and an addition portionof the solvent. After filtration and washing with solvent, the resin issuspended in an appropriate solvent such as DMF. A solution of about 2-3equivalents of an appropriately substituted halide 11b and a suitablebase such DIEA is added to the resin bound amino acid and this mixtureis shaken for 18-36 h. The resulting mixture is washed with severalportions of a suitable solvent and is suspended and shaken in an acidicsolution, such as 50% TFA/CH₂Cl₂, over several hours to cleave the acidfrom the resin to give a mixture of the Nα,Nα-bis-cinnamyl amino acid11c and the Nα-cinnamyl amino acid 11d.

[0064] By varying the resin bound amino acid 11a, one may obtain many ofthe compounds of the invention. The following resin bound amino acidsmay be used in Scheme 11: alanine,N-g-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)arginine,β-(4-methyltrityl)asparagine, aspartic acid (β-t-butyl ester),S-(trityl)cysteine, γ-(4-methyltrityl)glutamine, glutamic acid(β-t-butyl ester), glycine, N-imidazolyl-(trityl)histidine, isoleucine,leucine, N-ε-(2-chlorobenzyloxycarbonyl)lysine,N-ε-(t-butoxycarbonyl)lysine, methionine, phenylalanine, proline,O-(t-butyl)serine, O-(t-butyl)threonine,N-indolyl-(t-butoxycarbonyl)tryptophan, O-(t-butyl)tyrosine, valine,β-alanine, α-aminoadipic acid, 4-aminobutanoic acid,, 6-aminohexanoicacid, α-aminosuberic acid, 5-aminopentanoic acid, p-aminophenylalanine,α-aminopimelic acid γ-carboxyglutamic acid, p-carboxyphenylalanine,carnitine, citrulline, α,β-diaminopropionic acid, α,γ-diaminobutyricacid, homocitrulline, homoserine, and statine.

Scheme 12, Step A

[0065] An equivalent of an N-Fmoc-protected amino acid which is bound toa resin 11a is suspended in a suitable solvent such as DMF. This solventis removed and the nitrogen protecting group (Fmoc) is removed bystirring the resin bound amino acid with an organic base, such aspiperidine, and an addition portion of the solvent. After filtration andwashing with solvent, the resin is suspended in an appropriate solventsuch as trimethyl orthoformate (TMOF), an appropriately substitutedaldehyde 12a (5 equivalents) is added, and the mixture is shaken underN₂ overnight. This mixture is treated with a suspension of NaBH(OAc)₃ (5equivalents) in CH₂Cl₂ and shaken under N₂ overnight. After filtrationand washing with a suitable solvent, the resulting product, resin boundNα-monosubstituted amino acid 12b, is suspended and shaken in an acidicsolution, such as 50% TFA/CH₂Cl₂, over several hours to cleave the acidfrom the resin to give the Nα-cinnamyl amino acid 11 d.

Scheme 12, Step B

[0066] The resin 12b is suspended in an appropriate solvent such as DMFand is filtered. The appropriately substituted halide 12c and anappropriate base such as DIEA are added with some additional solvent andthe mixture is shaken under N₂ for 18-36 h. The resin boundNα,Nα-cinnamyl amino acid 12d is isolated from the suspension and theresin is cleaved with an acidic solution as described above to give thefree acid 12e.

General Procedure for the Solution-phase Synthesis of Nα,Nα-Bis-CinnamylAmino Acids and Nα-Cinnamyl Amino Acids Scheme 13

[0067] A solution of of amino acid ester 13a, an appropriatelysubstituted halide 11b, and an appropriate base such as DIEA, Na₂CO₃, orCs₂CO₃ in a suitable solvent, such as DMF, is heated at 50-100° C. underN₂ overnight, or until the starting material is exhausted, to give amixture of the Nα,Nα-bis-cinnamyl amino acid ester 13b and Nα-cinnamylamino acid ester 13c. If the side chain of R¹ contains an acid-cleavableprotecting group such as t-butylcarbamate, t-butyl ester, or t-butylether, those groups may be cleaved by treatment with an acidic solutionsuch as 30-80% TFA/CH₂Cl₂ or 2-4N HCl in EtOAc. For examples of 13b and13c where the ester group R⁴ is a primary alkyl group such as methyl orethyl, esters 13b and 13c may be independently converted to thecorresponding acids 11c and 11d by hydrolysis with an appropriate basesuch as aqueous NaOH, KOH, or LiOH. For examples of 13b and 13c wherethe ester group R⁴ is an acid-cleavable group such as t-butyl, esters13b and 13c may be independently converted to the corresponding acids11c and 11d by treatment with an acidic solution such as 30-80%TFA/CH₂Cl₂ or 2-4N HCl in EtOAc.

Scheme 14, Step A

[0068] A solution of 1 mmol of amino acid ester and 1-1.5 mmol of theappropriately substituted aldehyde 12a in 3-5 mL of TMOF was stirred atroom temperature under N₂ overnight. The solution was concentrated andused directly for the next reaction; optionally, the solution waspartitioned between EtOAc and water, washed with brine, dried overNa₂SO₄, and concentrated to give crude product, which was purified byMPLC to give mono-substituted product 14a. For examples of 14a whereinthe side chain R¹ contained an acid-cleavable protecting group such ast-butylcarbamate, t-butyl ester, or t-butyl ether, 8c was treated withan acidic solution such as 30-80% TFA/CH₂Cl₂ or 2-4N HCl in EtOAc. Thereaction mixture was concentrated and optionally dissolved in HOAc andfreeze-dried to give the deprotected form of 14a. For examples of 14awhere the ester group R⁴ is a primary alkyl group such as methyl orethyl, esters 14a may be converted to the corresponding acids 11d byhydrolysis with an appropriate base such as aqueous NaOH, KOH, or LiOH.For examples of 14a where the ester group R⁴ is an acid-cleavable groupsuch as t-butyl, esters 14a may be converted to the corresponding acids11d by treatment with an acidic solution such as 30-80% TFA/CH₂Cl₂ or2-4N HCl in EtOAc.

Scheme 14, Step B

[0069] Amino ester 14a was dissolved in DMF, combined with 1.1-1.5 mmolof the appropriately substituted chloride or bromide 12c, and heated at50-100° C. overnight. The reaction mixture was cooled and partitionedbetween water and EtOAc. The organic layer was washed with water andbrine, dried over Na₂SO₄, and concentrated. The crude product waspurified by MPLC to give pure 14b. For examples of 14b wherein the sidechain R¹ contained an acid-cleavable protecting group such ast-butylcarbamate, t-butyl ester, or t-butyl ether, 8c was treated withan acidic solution such as 30-80% TFA/CH₂Cl₂ or 2-N HCl in EtOAc. Thereaction mixture was concentrated and optionally dissolved in HOAc andfreeze-dried to give the deprotected form of 14b. For examples of 14bwhere the ester group R⁴ is a primary alkyl group such as methyl orethyl, esters 14b may be converted to the corresponding acids 12e byhydrolysis with an appropriate base such as aqueous NaOH, KOH, or LiOH.For examples of 14b where the ester group R⁴ is an acid-cleavable groupsuch as t-butyl, esters 14b may be converted to the corresponding acids12e by treatment with an acidic solution such as 30-80% TFA/CH₂Cl₂ or2-4N HCl in EtOAc.

[0070] Although the claimed compounds are useful as competitive bindersto the EPO receptor, some compounds are more active than others and areeither preferred or particularly preferred.

[0071] The particularly preferred “R¹” s are the side chain of lysine,ornithine, arginine, aspartic acid, glutamic acid, glutamine, cysteine,methionine, serine, and threonine.

[0072] The particularly preferred “R² and R³” s are phenoxy, substitutedphenoxy, benzyloxy, and substituted benzyloxy.

[0073] The particularly preferred “R⁴ and R⁵” s are phenoxy, substitutedphenoxy, benzyloxy, and substituted benzyloxy.

[0074] The particularly preferred “W” is —CH═CH—

[0075] The particularly preferred “Q” is —CH═CH—

[0076] The particularly preferred “X” are C₁₋₅alkenyl and CH₂.

[0077] The particularly preferred “Y” are C₁₋₅alkenyl and CH₂.

[0078] The particularly preferred “n” are 1 and 2.

[0079] The particularly preferred “Z” are hydroxy, methoxy,phenethylamino, substituted phenethylamino, and—NH(CH₂)₂O(CH₂)₂O(CH₂)₂NH—.

[0080] Pharmaceutically useful compositions the compounds of the presentinvention, may be formulated according to known methods such as by theadmixture of a pharmaceutically acceptable carrier. Examples of suchcarriers and methods of formulation may be found in Remington'sPharmaceutical Sciences. To form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of the compound of the presentinvention.

[0081] Therapeutic or diagnostic compositions of the invention areadministered to an individual in amounts sufficient to treat or diagnosedisorders in which modulation of EPO receptor-related activity isindicated. The effective amount may vary according to a variety offactors such as the individual's condition, weight, sex and age. Otherfactors include the mode of administration. The pharmaceuticalcompositions may be provided to the individual by a variety of routessuch as subcutaneous, topical, transdermal, oral and parenteral.

[0082] The term “chemical derivative” describes a molecule that containsadditional chemical moieties which are not normally a part of the basemolecule. Such moieties may improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties mayattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Examples of such moieties are describedin a variety of texts, such as Remington's Pharmaceutical Sciences.

[0083] Compounds disclosed herein may be used alone at appropriatedosages defined by routine testing in order to obtain optimal inhibitionof the EPO receptor or its activity while minimizing any potentialtoxicity. In addition, co-administration or sequential administration ofother agents may be desirable.

[0084] The present invention also has the objective of providingsuitable topical, transdermal, oral, systemic and parenteralpharmaceutical formulations for use in the novel methods of treatment ofthe present invention. The compositions containing compounds accordingto this invention as the active ingredient for use in the modulation ofEPO receptors can be administered in a wide variety of therapeuticdosage forms in conventional vehicles for administration. For example,the compounds or modulators can be administered in such oral dosageforms as tablets, capsules (each including timed release and sustainedrelease formulations), pills, powders, granules, elixirs, tinctures,solutions, suspensions, syrups and emulsions, or by transdermal deliveryor injection. Likewise, they may also be administered in intravenous(both bolus and infusion), intraperitoneal, subcutaneous, topical withor without occlusion, transdermal, or intramuscular form, all usingforms well known to those of ordinary skill in the pharmaceutical arts.The compounds of the present invention may be delivered by a widevariety of mechanisms, including but not limited to, transdermaldelivery, or injection by needle or needle-less injection means. Aneffective but non-toxic amount of the compound desired can be employedas an EPO receptor modulating agent.

[0085] The daily dosage of the products may be varied over a wide rangefrom 0.01 to 1,000 mg per patient, per day. For oral administration, thecompositions are preferably provided in the form of scored or unscoredtablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0,25.0, and 50.0 milligrams of the active ingredient for the symptomaticadjustment of the dosage to the patient to be treated. An effectiveamount of the drug is ordinarily supplied at a dosage level of fromabout 0.0001 mg/kg to about 100 mg/kg of body weight per day. The rangeis more particularly from about 0.001 mg/kg to 10 mg/kg of body weightper day. The dosages of the EPO receptor modulators are adjusted whencombined to achieve desired effects. On the other hand, dosages of thesevarious agents may be independently optimized and combined to achieve asynergistic result wherein the pathology is reduced more than it wouldbe if either agent were used alone.

[0086] Advantageously, compounds or modulators of the present inventionmay be administered in a single daily dose, or the total daily dosagemay be administered in divided doses of two, three or four times daily.Furthermore, compounds or modulators for the present invention can beadministered in intranasal form via topical use of suitable intranasalvehicles, or via transdermal routes, using those forms of transdermalskin patches well known to those of ordinary skill in that art. To beadministered in the form of a transdermal delivery system, the dosageadministration will, of course, be continuous rather than intermittentthroughout the dosage regimen.

[0087] For combination treatment with more than one active agent, wherethe active agents are in separate dosage formulations, the active agentscan be administered concurrently, or they each can be administered atseparately staggered times.

[0088] The dosage regimen utilizing the compounds or modulators of thepresent invention is selected in accordance with a variety of factorsincluding type, species, age, weight, sex and medical condition of thepatient; the severity of the condition to be treated; the route ofadministration; the renal and hepatic function of the patient; and theparticular compound thereof employed. A physician or veterinarian ofordinary skill can readily determine and prescribe the effective amountof the drug required to prevent, counter or arrest the progress of thecondition. Optimal precision in achieving concentrations of drug withinthe range that yields efficacy without toxicity requires a regimen basedon the kinetics of the drug's availability to target sites. Thisinvolves a consideration of the distribution, equilibrium, andelimination of a drug.

[0089] In the methods of the present invention, the compounds ormodulators herein described in detail can form the active ingredient,and are typically administered in admixture with suitable pharmaceuticaldiluents, excipients or carriers (collectively referred to herein as“carrier” materials) suitably selected with respect to the intended formof administration, that is, oral tablets, capsules, elixirs, syrups andthe like, and consistent with conventional pharmaceutical practices.

[0090] For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Moreover, when desired or necessary,suitable binders, lubricants, disintegrating agents and coloring agentscan also be incorporated into the mixture. Suitable binders include,without limitation, starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include, without limitation, sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum and the like.

[0091] For liquid forms the active drug component can be combined insuitably flavored suspending or dispersing agents such as the syntheticand natural gums, for example, tragacanth, acacia, methyl-cellulose andthe like. Other dispersing agents which may be employed include glycerinand the like. For parenteral administration, sterile suspensions andsolutions are desired. Isotonic preparations which generally containsuitable preservatives are employed when intravenous administration isdesired.

[0092] Topical preparations containing the active drug component can beadmixed with a variety of carrier materials well known in the art, suchas, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and Eoils, mineral oil, PPG2 myristyl propionate, and the like, to form,e.g., alcoholic solutions, topical cleansers, cleansing creams, skingels, skin lotions, and shampoos in cream or gel formulations.

[0093] The compounds or modulators of the present invention can also beadministered in the form of liposome delivery systems, such as smallunilamellar vesicles, large unilamellar vesicles and multilamellarvesicles. Liposomes can be formed from a variety of phospholipids, suchas cholesterol, stearylamine or phosphatidylcholines.

[0094] Compounds of the present invention may also be delivered by theuse of monoclonal antibodies as individual carriers to which thecompound molecules are coupled. The compounds or modulators of thepresent invention may also be coupled with soluble polymers astargetable drug carriers. Such polymers can includepolyvinyl-pyrrolidone, pyran copolymer,polyhydroxypropylmethacryl-amidephenol,polyhydroxy-ethylaspartamidephenol, or polyethyl-eneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds ormodulators of the present invention may be coupled to a class ofbiodegradable polymers useful in achieving controlled release of a drug,for example, polylactic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydro-pyrans,polycyanoacrylates and cross-linked or amphipathic block copolymers ofhydrogels, and other suitable polymers known to those skilled in theart.

[0095] For oral administration, the compounds or modulators may beadministered in capsule, tablet, or bolus form or alternatively they canbe mixed in the animals feed. The capsules, tablets, and boluses arecomprised of the active ingredient in combination with an appropriatecarrier vehicle such as starch, talc, magnesium stearate, or di-calciumphosphate. These unit dosage forms are prepared by intimately mixing theactive ingredient with suitable finely-powdered inert ingredientsincluding diluents, fillers, disintegrating agents, and/or binders suchthat a uniform mixture is obtained. An inert ingredient is one that willnot react with the compounds or modulators and which is non-toxic to theanimal being treated. Suitable inert ingredients include starch,lactose, talc, magnesium stearate, vegetable gums and oils, and thelike. These formulations may contain a widely variable amount of theactive and inactive ingredients depending on numerous factors such asthe size and type of the animal species to be treated and the type andseverity of the infection. The active ingredient may also beadministered as an additive to the feed by simply mixing the compoundwith the feedstuff or by applying the compound to the surface of thefeed. Alternatively the active ingredient may be mixed with an inertcarrier and the resulting composition may then either be mixed with thefeed or fed directly to the animal. Suitable inert carriers include cornmeal, citrus meal, fermentation residues, soya grits, dried grains andthe like. The active ingredients are intimately mixed with these inertcarriers by grinding, stirring, milling, or tumbling such that the finalcomposition contains from 0.001 to 5% by weight of the activeingredient.

[0096] The compounds or modulators may alternatively be administeredparenterally via injection of a formulation consisting of the activeingredient dissolved in an inert liquid carrier. Injection may be eitherintramuscular, intraruminal, intratracheal, or subcutaneous, either byneedle or needle-less means. The injectable formulation consists of theactive ingredient mixed with an appropriate inert liquid carrier.Acceptable liquid carriers include the vegetable oils such as peanutoil, cotton seed oil, sesame oil and the like as well as organicsolvents such as solketal, glycerol formal and the like. As analternative, aqueous parenteral formulations may also be used. Thevegetable oils are the preferred liquid carriers. The formulations areprepared by dissolving or suspending the active ingredient in the liquidcarrier such that the final formulation contains from 0.005 to 10% byweight of the active ingredient.

[0097] Topical application of the compounds or modulators is possiblethrough the use of a liquid drench or a shampoo containing the instantcompounds or modulators as an aqueous solution or suspension. Theseformulations generally contain a suspending agent such as bentonite andnormally will also contain an antifoaming agent. Formulations containingfrom 0.005 to 10% by weight of the active ingredient are acceptable.Preferred formulations are those containing from 0.01 to 5% by weight ofthe instant compounds or modulators.

[0098] The compounds of Formula I may be used in pharmaceuticalcompositions to treat patients (humans and other mammals) with disordersor conditions associated with the production of erythropoietin ormodulated by the EPO receptor. The compounds can be administered in themanner of the commercially available product or by any oral orparenteral route (including but not limited to, intravenous,intraperitoneal, intramuscular, subcutaneous, dermal patch), where thepreferred route is by injection. When the method of administration isintravenous infusion, compound of Formula I may be administered in adose range of about 0.01 to 1 mg/kg/min. For oral administration, thedose range is about 0.1 to 100 mg/kg.

[0099] The pharmaceutical compositions can be prepared usingconventional pharmaceutical excipients and compounding techniques. Oraldosage forms may be used and are elixirs, syrups, capsules, tablets andthe like. Where the typical solid carrier is an inert substance such aslactose, starch, glucose, methyl cellulose, magnesium stearate,dicalcium phosphate, mannitol and the like; and typical liquid oralexcipients include ethanol, glycerol, water and the like. All excipientsmay be mixed as needed with disintegrants, diluents, granulating agents,lubricants, binders and the like using conventional techniques known tothose skilled in the art of preparing dosage forms. Parenteral dosageforms may be prepared using water or another sterile carrier.

[0100] Typically the compounds of Formula I are isolated as the freebase, however when possible pharmaceutically acceptable salts can beprepared. Examples of such salts include hydrobromic, hydroiodic,hydrochloric, perchloric, sulfuric, maleic, fumaric, malic, tartaric,citric, benzoic, mandelic, methanesulfonic, hydroethanesulfonic,benzenesulfonic, oxalic, pamoic, 2-naphthalenesulfonic,p-toluenesulfonic, cyclohexanesulfamic and saccharic.

[0101] In order to illustrate the invention the following examples areincluded. These examples do not limit the invention. They are only meantto suggest a method of practicing the invention. Those knowledgeable inchemical synthesis and the treatment of EPO related disorders may findother methods of practicing the invention. However those methods aredeemed to be within the scope of this invention.

BIOLOGICAL EXAMPLES

[0102] The compounds of the invention were evaluated for the ability tocompete with EPO in the following immobilized EPO receptor preparation(EBP-Ig, EPO binding protein-Ig).

[0103] EBP-Ig fusion protein (as disclosed in WO97/27219 which is hereinincorporated by reference) was purified by affinity chromatography fromthe conditioned media of NSO cells engineered to express a recombinantgene construct which functionally joined the N-terminal 225 amino acidsof the human EPO receptor and an Ig heavy chain as described herein. Theinteraction of biotin and streptavidin is frequently employed to captureand effectively immobilize reagents useful in assay protocols and hasbeen employed here as a simple method to capture and immobilize EBP-Ig.EBP-Ig is initially randomly modified with an amine reactive derivativeof biotin to produce biotinylated-EBP-Ig. Use of streptavidin coatedplates allows the capture of the biotinylated EBP-Ig on the surface of ascintillant impregnated coated well (Flash plates, NEN-DuPont). Uponbinding of [¹²⁵I]EPO to the ligand binding domain, specific distancerequirements are satisfied and the scintillant is induced to emit lightin response to the energy emitted by the radioligand. Unboundradioligand does not produce a measurable signal because the energy fromthe radioactive decay is too distant from the scintillant. The amount oflight produced was quantified to estimate the amount of ligand binding.The specific assay format was suitable for the multi-well plate capacityof a Packard TopCount Microplate Scintillation counter. Compounds whichwere capable of reducing the amount of detected signal throughcompetitive binding with the radioligand were identified.

[0104] Biotinylated EBP-Ig was prepared as follows. EBP-Ig (3 mL, OD₂₈₀2.9) was exchanged into 50 mM sodium bicarbonate, pH 8.5 using aCentriprep 10 ultrafiltration device. The final volume of the exchangedprotein was 1.2 mL (OD₂₈₀ 2.6, representing about 2 mg total protein).10 μL of a 4 mg/ml solution of NHS-LC-Biotin (Pierce) was added and thereaction mixture placed on ice in the dark for two hours. Unreactedbiotin was removed by exchange of the reaction buffer into PBS in aCentriprep 10 device and the protein reagent aliquoted and stored at−70° C.

[0105] Each individual binding well (200 μL) contained finalconcentrations of 1 μg/mL of biotinylated EBP-Ig, 0.5 nM of [¹²⁵I]EPO(NEN Research Products, Boston, 100 μCi/μg) and 0-500 μM of testcompound (from a 10-50 mM stock in 100% DMSO). All wells were adjustedto a final DMSO concentration of 5%. All assay points were performed intriplicate and with each experiment a standard curve for unlabelled EPOwas performed at final concentration of 2000, 62, 15, 8, 4, and 0 nM.After all additions were made, the plate was covered with an adhesivetop seal and placed in the dark at room temperature overnight. The nextday all liquid was aspirated from the wells to limit analyte dependentquench of the signal, and the plates were counted on a Packard TOPCOUNTMicroplate Scintillation Counter. Non-specific binding (NSB) wascalculated as the mean CPM of the 2000 nM EPO wells and total binding(TB) as the mean of the wells with no added unlabelled EPO. Correctedtotal binding (CTB) was calculated as: TB−NSB=CTB. The concentration oftest compound which reduced CTB to 50% was reported as the IC₅₀.Typically the IC₅₀ value for unlabelled EPO was ca. 2-7 nM and EMP1 was0.1 μM. Table 1 lists the average % inhibition, and if determined theIC₅₀ and IC₃₀ values for compounds of Formula I, where the compoundnumbers refer to the compounds in the tables accompanying thepreparative examples. TABLE 1 Inhibition of EPO binding to EBP-Ig cpd %inh @ 50 μM IC₃₀, μM* IC₅₀, μM*  11 70 nd nd  12 59 nd nd  14 30 nd nd 15 48 nd nd  77 52 30 40  82 32 nd nd  86 37 nd nd 100 34 nd nd 101 32nd nd 104 78 10 30 105 70 25 35 107 78 30 42 108 81 23 36 110 54 6 10112 59 2 10 114 37 10 nd 115 35 nd nd 116 32 nd nd 117 34 nd nd 118 36 210 119 34 nd nd 120 35 nd nd 121 45 6 nd 137 60 5 30 139 46 2 10 178 36nd nd 179 30 nd nd 183 36 nd nd 184 53 10 nd 203 37 50 nd 211 62 20 65220 45 30 50 221 48 10 80 222 56 5 nd 224 51 25 nd 227 48 20 50 230 42nd nd 231 36 nd nd 235 49 20 50 237 55 30 70 238 39 nd nd 239 46 8 50243 75 2 18 244 66 1 28 246 79 10 75 247 47 7 18 248 56 7 20 249 72 7 10250 78 7 20 251 49 10 45 261 51 1.5 2 262 93 1 1.5 263 88 1 1.5 264 891.5 8 265 65 1 6 266 82 1 4 267 83 2 6 268 40 nd nd 269 55 8 85 270 56 7100 271 77 2 7 272 78 5 10 285 41 nd nd 286 46 35 65 287 36 nd nd 300 5735 145 305 48 35 225 312 45 10 85 321 42 45 nd 363 33 35 220 366 38 65nd 368 40 90 nd

PREPARATIVE EXAMPLES

[0106] Unless otherwise noted, materials used in the examples wereobtained from commercial suppliers and were used without furtherpurification. Melting points were determined on a Thomas Hooverapparatus and are uncorrected. Proton nuclear magnetic resonance (¹HNMR) spectra were measured in the indicated solvent withtetramethylsilane (TMS) as the internal standard using a Bruker AC-300NMR spectrometer. NMR chemical shifts are expressed in parts per million(ppm) downfield from internal TMS using the d scale. ¹H NMR data aretabulated in order: multiplicity, (s, singlet; d, doublet; t, triplet;q, quartet; m, multiplet), number of protons, coupling constant inHertz). Electrospray (ES) mass spectra (MS) were determined on a HewlettPackard Series 1090 LCMS Engine. Elemental analyses were performed byQuantitative Technologies, Inc. (QTI), PO Box 470, Salem IndustrialPark, Bldg #5, Whitehouse, N.J. 08888-0470. Analytical thin layerchromatography (TLC) was done with Merck Silica Gel 60 F₂₅₄ plates (250micron). Medium pressure liquid chromatography (MPLC) was done withMerck Silica Gel 60 (230-400 mesh).

Example 1 N,N-bis(3-Phenoxycinnamyl)Glu(O-t-Bu)-OMe (cpd 96) andN-(3-phenoxycinnamyl)Glu(O-t-Bu)-OMe (cpd 334)

[0107] A solution of 500 mg (1.97 mmol) of H-Glu(O-t-Bu)OMe.HCl, 997 mg(3.45 mmol) of 3-phenoxycinnamyl bromide (Jackson, W. P.; Islip, P. J.;Kneen, G.; Pugh, A.; Wates, P. J. J.Med.Chem. 31 1988; 499-500), and0.89 mL (5.1 mmol, 660 mg) of DIEA in 5 mL of DMF was stirred under N₂at room temperature for 40 h. The mixture was partitioned between EtOAcand water and the organic layer was washed with water and brine. Afterdrying over Na₂SO₄, the organic solution was concentrated to give 1.24 gof orange oil. The crude residue was purified by MPLC using a solventgradient ranging from 10-30% EtOAc/hexanes to give two products. Theless polar product (cpd 96, 235 mg, 19% based on starting amino acid),was isolated as a pale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.39 (s, 9H),2.0 (m, 2H), 2.33 (dt, 2H, J=2, 7 Hz), 3.24 (dd, 2H, J=8, 15 Hz), 3.5,(m, 3H), 3.69 (s, 3H), 6.13 (m, 2H), 6.47 (d, 2H, J=16 Hz), 6.86 (dd,2H, J=1.5, 8 Hz), 7.0-7.4 (complex, 16H); MS (ES+) m/z 634 (MH+).

[0108] The more polar product (cpd 334, 422 mg, 50% based on startingamino acid) was isolated as a pale yellow oil; ¹H NMR (CDCl₃, 300 MHz)1.42 (s, 9H), 1.9 (m, 2H), 2.35 (t, 2H, J=7.5 Hz), 3.2-3.4 (complex,3H), 3.71 (s, 3H), 6.17 (dt, 1H, J=16, 6 Hz), 6.46 (d, 1H, J=16 Hz),6.87 (dd, 1H, J=1.5, 8 Hz), 7.01 (m, 3H), 7.10 (t, 2H, J=7.5 Hz),7.2-7.4 (complex, 3H); MS (ES+) m/z 426 (MH+). Anal. Calcd forC₂₅H₃₁NO₅: C, 70.57; H, 7.34; N, 3.29. Found: C, 70.29; H, 7.14; N,3.08.

Example 2 N-(3-Phenoxycinnamyl)Glu-OMe (cpd 325)

[0109] A solution of 95 mg (0.22 mmol) ofN-(3-phenoxycinnamyl)Glu(O-t-Bu)-OMe (cpd 334) in 3 mL of 50% TFA/CH₂Cl₂was stirred for 2 h at room temperature. The mixture was concentratedand the residue was dissolve in acetic acid and freeze-dried to give 117mg of N-(3-phenoxycinnamyl)Glu-OMe (cpd 325)as an off-white solid; ¹HNMR (CD₃OD, 300 MHz) 2.3-2.7 (complex, 4H), 3.78 (s, 3H), 3.81 (d, 2H,J=7 Hz), 4.09 (t, 1H, J=5 Hz), 6.17 (dt, 1H, J=16, 7 Hz), 6.55 (d, 1H,J=16 Hz), 6.9 (m, 4H), 7.11 (t, 2H, J=7.5 Hz), 7.3 (m, 4H); MS (ES+) m/z370 (MH+), 209. Anal. Calcd for C₂₁H₂₃NO₅.C₂HF₃O₂: C, 57.14; H, 5.00; N,2.90. Found: C, 57.07; H, 5.02; N, 2.73.

Example 3 N,N-bis(3-Phenoxycinnamyl)Asp(O-t-Bu)-O-t-Bu (cpd 106)

[0110] A solution of 1.00 g (3.55 mmol) of Asp(O-t-Bu)-O-t-Bu.HCl, 2.05g (7.1 mmol) of 3-phenoxycinnamyl bromide, and 1.86 mL (10.7 mmol, 1.38g) of DIEA in 15 mL of DMF was heated under N₂ at 60° C. overnight.Additional 3-phenoxycinnamyl bromide (1.0 g, 3.4 mmol) and DIEA (0.95mL, 0.705 g, 5.4 mmol) were added and heating was continued for anadditional 14 h. The mixture was cooled and partitioned between EtOAcand water. The organic layer was washed twice with water, once withbrine, and was dried over Na₂SO₄. The solution was concentrated to give3.5 g of an amber oil which was purified by MPLC using a solventgradient ranging from 2.5-3% EtOAc/hexanes to afford 1.21 g of cpd 106as a pale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.41 (s, 9H), 1.48 (s,9H), 2.49 (dd, 1H, J=7.5, 15.5 Hz), 2.70 (dd, 1H, J=7.5, 15.5 Hz), 3.26(dd, 2H, J=7.5, 14.5 Hz), 3.47 (dd, 2H, J=4, 14.5 Hz), 3.88 (t, 1H,J=7.5), 6.13 (m, 2H), 6.48 (d, 2H, J=16 Hz), 6.86 (dd, 2H, J=2, 8 Hz),7.0 (m, 6H), 7.1 (m, 4H), 7.2-7.4 (complex, 6H); MS (ES+) m/z 662 (MH+).

Example 4 N,N-bis(3-Phenoxycinnamyl)Asp-OH (cpd 107)

[0111] A solution of 1.14 g (1.62 mmol) of cpd 106 in 16 mL of 50%TFA/CH₂Cl₂ was stirred at room temperature for 24 h. The solution wasconcentrated and pumped to give 1.37 g (˜100%) cpd 107 as an amber oil;¹H NMR (CD₃OD, 300 MHz) 3.1 (m, 2H), 4.0 (dd, 2H, J=8, 14 Hz), 4.27 (dd,2H, J=8, 16 Hz), 4.70 (t, 1H, J=4 Hz), 6.38 (2H, dt, J=16, 8 Hz),6.7-7.4 (complex, 20H); MS (ES−) m/z 562 ([M-H]+).

Example 5 N,N-bis(4-Benzyloxybenzyl)Lys(Boc)-OMe (cpd 111) andN-(4-Benzyloxybenzyl)Lys(Boc)-OMe

[0112] A solution of 594 mg (2.0 mmol) of Lys(Boc)-OMe.HCl, 524 mg (2.25mmol) of 4-benzyloxybenzyl chloride, 75 mg (0.5 mmol), of NaI, and 0.61mL (3.5 mol, 452 mg) of DIEA was warmed at 50-70° C. under N₂ overnight.The mixture was cooled and partioned between EtOAc and water. Theorganic layer was washed twice with water, once with brine, and wasdried over Na₂SO₄. The organic solution was concentrated to give 0.83 gof amber oil which was purified by MPLC using a solvent gradient rangingfrom 15-40% EtOAc/hexanes to give two products. The less polar product(296 mg), cpd 111, was isolated as a pale yellow oil; ¹H NMR (CDCl₃, 300MHz) 1.28 (m, 4H), 1.43 (s, 9H), 1.70 (m, 2H), 3.03 (m, 2H), 3.28 (t,1H, J=7 Hz), 3.40 (d, 2H, J=13.5 Hz), 3.74 (s, 3H), 3.81 (d, 2H, J=13.5Hz), 5.05 (2, 4H), 6.92 (d, 4H, J=8.5), 7.23 (d, 4H, J=8.5), 7.25-7.5(complex, 10H); MS (ES+) m/z 653 (MH+).

[0113] The more polar product (406 mg),N-(4-benzyloxybenzyl)Lys(Boc)-OMe, was isolated as a white solid; ¹H NMR(CDCl₃, 300 MHz) 1.4 (, 4H), 1.43 (s, 9H), 1.65 (m, 3H), 3.08 (m, 2H),3.23 (t, 1H, J=6.5 Hz), 3.54 (d, 1H, J=12.5 Hz), 3.71 (s, 3H), 3.73 (d,1H, J=12.5 Hz), 5.05 (s, 2H), 6.92 (d, 2H, J=8.5 Hz), 7.23 (d, 2H, J=8.5Hz), 7.25-7.5 (complex, 5H); MS (ES+) m/z 457 (MH+).

Example 6 N-(4-Benzyloxybenzyl)-N-(3-nitrobenzyl)Lys(Boc)-OMe (cpd 113)

[0114] A solution of 374 mg (0.82 mmol) ofN-(4-Benzyloxybenzyl)Lys(Boc)-OMe, 221 mg (1.03 mmol) of 4-nitrobenzylbromide, and 197 L (1.13 mmol, 146 mg) of DIEA was warmed at 50-70° C.for 4 h, then at 40-50° C. overnight. After the addition of 0.2 mL of 1Naqueous HCl, the mixture was partioned between EtOAc and water. Theorganic layer was washed twice with water, once with brine, and wasdried over Na₂SO₄. The organic solution was concentrated to give 610 mgof an amber oil which was purified by MPLC 1:3 EtOAc/hexanes to afford436 mg (90%) cpd 113 as a pale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.35(m, 4H), 1.42 (s, 9H), 1.75 (broad q, 2H, J=8 Hz), 3.06 (broad q, 2H,J=6 Hz), 3.28 (t, 1H, J=7.5 Hz), 3.48 (d, 1H, J=13.5 Hz), 3.66 (d, 1H,J=14.5 Hz), 3.76 (s, 3H), 3.79 (d, 1H, J=13.5 Hz), 3.97 (d, 1H, J=14.5Hz), 4.47 (broad s, 1H), 5.05 (s, 2H), 6.93 (d, 2H, J=8.5 Hz), 7.22 (d,2H, J=8.5 Hz), 7.3-7.5 (complex, 6H), 7.65 (d, 1H, J=7.5 Hz), 8.09 (d,1H, J=8 Hz), 8.22 (s, 1H); MS (ES+) m/z 592 (MH+).

Example 7 N-(3-Aminobenzyl)-N-(4-benzyloxybenzyl)Lys(Boc)-OMe

[0115] A solution of 361 mg (0.61 mmol) of cpd 113 and 835 mg (3.7 mmol)of SnCl₂ dihydrate was stirred under N₂ at room temperature for 6 h. Theslightly cloudy mixture was poured into 200 mL of 5% aqueous Na₂CO₃ withrapid stirring. The resulting milky suspension was extracted with three75 mL portions of CH₂Cl₂ and the combined organic layers were washedwith brine and dried over Na₂SO₄. The extracts were concentrated to give344 mg of colorless oil which was purified by MPLC using 1:2EtOAc/hexanes to provide 291 mg ofN-(3-aminobenzyl)-N-(4-benzyloxybenzyl)Lys(Boc)-OMe as a yellow oil; ¹HNMR (CDCl₃, 300 MHz) 1.25 (m, 4H), 1.44 (s, 9H), 1.70 (m, 2H), 3.31 (dd,1H, J=6, 9 Hz), 3.38 (d, 1H, J=14 Hz), 3.40 (d, 1H, J=13.5 Hz), 3.74 (s,3H), 3.81 (d, 1H, J=14 Hz), 3.83 (d, 1H, J=13.5 Hz), 4.52 (broad s, 1H),5.05 (s, 2H), 6.50 (broad d, 1H, J=8 Hz), 6.70 (m, 2H), 6.92 (d, 2H,J=8.5 Hz), 7.08 (t, 1H, J=7.5 Hz), 7.2-7.5 (complex, 7H); MS (ES+) m/z562 (base, MH+), 506.

Example 8N-(4-Benzyloxybenzyl)-N-(3-((2-furancarbonyl)amino)benzyl)Lys-OMe (cpd117)

[0116] A solution of 42 mg (0.075 mmol) ofN-(3-aminobenzyl)-N-(4-benzyloxybenzyl)Lys(Boc)-OMe and 12 μL (12 mg,0.15 mmol) of pyridine in 0.5 mL of 1,2-dichloroethane was combined with8.1 μL (11 mg, 0.083 mmol) and stirred under N₂ overnight. EtOAc (3 mL)was added and the solution was washed twice with 2 mL of water and 2 mLof saturated aqueous NaHCO₃. The EtOAc solution was filtered through apad of Na₂SO₄ and concentrated to give 44 mg ofN-(4-benzyloxybenzyl)-N-(3-((2-furancarbonyl)amino)benzyl)Lys(Boc)-OMe;MS (ES+) m/z 356 (MH+). The Boc-protected intermediate was stirred in 2mL of 50% TFA/CH₂Cl₂ for 2 h and was concentrated and pumped at highvacuum to provide 66 mg of cpd 117 as the bis-TFA salt; ¹H NMR (CD₃OD,300 MHz) 1.55 (m, 2H), 1.65 (m, 2H), 2.10 (m, 2H), 2.93 (t, 2H, J=7 Hz),3.68 (t, 1H, J=7 Hz), 3.78 (s, 3H), 4.20 (m, 4H), 5.09 (s, 2H), 6.66(dd, 1H, J=1.5, 3.5 Hz), 7.03 (d, 2H, J=8.5 Hz), 7.1-7.6 (complex, 11H),7.76 (m, H), 8.07 (m, 1H); MS (ES+) m/z 556 (base, MH+), 360, 197.

Example 9 N,N-bis(3-Nitrobenzyl)Asp(O-t-Bu)-O-t-Bu (cpd 62)

[0117] A solution of 0.50 mg (1.77 mmol) of Asp(O-t-Bu)-O-t-Bu.HCl, 1.17g (5.42 mmol) of 3-nitrobenzyl bromide, and 1.25 mL (0.93 g, 7.2 mmol)of DIEA in 6 mL of DMF was stirred at room temperature under N₂ for 24 hand was heated at 70-80° C. overnight. The reaction mixture waspartitioned between EtOAc and water and the organic layer was washedtwice with water and once with brine. After drying over Na₂SO₄, theorganic solution was concentrated to give 0.86 g of a yellow oil whichwas purified by MPLC using 1:9 EtOAc/hexanes to afford 0.849 g (93%) cpd62 as a pale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.43 (s, 9H), 1.57 (s,9H), 2.59 (dd, 1H, J=7.5, 16 Hz), 2.76 (dd, 1H, J=7, 16 Hz), 3.72 (t,1H, J=7.5 Hz), 3.78 (d, 2H, J=14 Hz), 3.92 (d, 2H, J=14 Hz), 7.47 (t,2H, J=8 Hz), 7.67 (d, 2H, J=7.5 Hz), 8.09 (broad d, 2H J=8 Hz), 8.16(broad s, 2H); MS (ES+) m/z 538 (MNa+), 516 (base, MH+), 460, 404, 237.

Example 10 N,N-bis(3-Aminobenzyl)Asp(O-t-Bu)-O-t-Bu

[0118] A solution of 0.644 g (1.25 mmol) of cpd 62 and 2.82 g (12.5mmol) of SnCl₂.2 H₂O in 12 mL of absolute EtOH was refluxed for 1.5 h.The mixture was cooled and poured into 300 mL of 5% aqueous Na₂CO₃ withrapid stirring. The cloudy mixture was extracted with three 150 mLportions of CH₂Cl₂ and the organic extracts were washed with brine anddried over Na₂SO₄. The CH₂Cl₂ solution was concentrated to afford 210 mg(37%) of N,N-bis(3-aminobenzyl)Asp(O-t-Bu)-O-t-Bu as a cloudy yellow oilwhich was used without purification; ¹H NMR (CDCl₃, 300 MHz) 1.40 (s,9H), 1.52 (s, 9H), 2.48 (dd, 1H, J=7, 16 Hz), 2.76 (dd, 1H, J=8, 16 Hz),3.48 (d, 2H, J=14 Hz), 3.55 (m, 1H), 3.73 (d, 2H, J=14 Hz), 6.56 (broadd, 2H J=8 Hz), 6.70 (broad s, 2H), 6.77 (d, 2H, J=7.5 Hz), 7.08 (t, 2H,J=8 Hz); MS (ES+) m/z 478 (MNa+), 456 (base, MH+), 400, 344.

Example 11 N,N-bis(3-(4-Methylbenzoyl)aminobenzyl)Asp(O-t-Bu)-O-t-Bu

[0119] To a solution of 109 mg (0.24 mmol) ofN,N-bis(3-aminobenzyl)Asp(O-t-Bu)-O-t-Bu, 29 mg (0.24 mmol) of DMAP, 125μL (93 mg, 0.72 mmol) of DIEA in 1 mL of CH₂Cl₂ was added 95 μL (111 mg,0.72 mmol) of 4-methylbenzoyl chloride. The solution was stirred underN₂ overnight and was then partitioned between EtOAc and water. Theorganic layer was washed with saturated aqueous NaHCO₃ and brine, driedover Na₂SO₄, and concentrated to give 177 mg of yellow oil. The crudematerial was purified by MPLC using a solvent gradient ranging from20-25% EtOAc/hexanes to provide 87 mg ofN,N-bis(3-(4-methylbenzoyl)aminobenzyl)Asp(O-t-Bu)-O-t-Bu as a paleyellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.36 (s, 9H), 1.55 (s, 9H), 2.35 (s,6H), 2.53 (dd, 1H, J=6, 16 Hz), 2.76 (dd, 1H, J=9, 16 Hz), 3.69 (d, 2H,J=14), 3.77 (dd, 1H, J=6, 9 Hz), 3.83 (d, 2H, J=14), 7.01 (m, 6H), 7.26(t, 2H, J=8 Hz), 7.59 (m, 6H), 8.11 (s, 2H), 8.49 (s, 2H); MS (ES+) m/z714 (MNa+), 692 (base, MH+), 636, 580.

Example 12 N,N-bis(3-(4-Methylbenzoyl)aminobenzyl)Asp-OH (cpd 64)

[0120] A solution of 87 mg (0.13 mmol) ofN,N-bis(3-(4-methylbenzoyl)amino-benzyl)Asp(O-t-Bu)-O-t-Bu in 1 mL of50% TFA/CH₂Cl₂ was stirred overnight. The mixture was concentrated andthe residue was dissolved in HOAc and freeze-dried to afford 77 mg cpd64 as a white solid; ¹H NMR (CD₃OD, 300 MHz) 2.40 (s, 6H), 2.85 (dd, 1H,J=6, 16.5 Hz), 2.98 (dd, 1H, J=8, 16.5 Hz), 4.02 (d, 2H, J=13.5 Hz),4.08 (d, 4H, J=13.5 Hz), 4.10 (t, 1H, J=6.5 Hz), 7.22 (m, 6H), 7.34 (t,2H, J=7.5 Hz), 7.60 (broad d, 2H, J=9 Hz), 7.76 (d, 4H, J=8 Hz), 7.88(broad s, 2H); MS (ES+) m/z 580 (base, MH+).

Example 13 [N-Cbz-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂

[0121] To a solution of 1.69 g (5.0 mmol) of N-Cbz-Glu(O-t-Bu)-OH, 0.365mL (0.371 g, 2.5 mmol) of 1,8-diamino-3,6-dioxaoctane, 0.743 g (5.5mmol) of HOBT, and 1.05 mL (0.776 g, 6.0 mmol) of DIEA in 15 mL ofCH₂Cl₂ was added 1.05 g (5.5 mmol) of EDCI in one portion. Afterstirring at room temperature under N₂ overnight, the mixture waspartitioned between EtOAc and 10% aqueous citric acid. The organic layerwas washed with water, saturated NaHCO₃, and brine, dried over Na₂SO₄,and concentrated to give 1.87 g of (N-Cbz-Glu(O-t-Bu)-NHCH₂CH₂OCH₂)₂ asa colorless oil; ¹H NMR (CD₃OD, 300 MHz) 1.43 (s, 18H), 1.85 (m, 2H),2.05 (m, 2H), 2.31 (t, 4H, J=8 Hz), 3.37 (t, 4H, J=5 Hz), 3.52 (t, 4H,J=5 Hz), 3.58 (s, 4H), 4.15 (m, 2H), 5.09 (dd, 4H, J=12, 16 Hz), 7.30(m, 10H); MS (ES+) m/z 809 (base, MNa+), 787 (MH+).

Example 14 [Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂

[0122] Ammonium formate (0.78 g, 12.4 mmol) and 0.16 g of 10% palladiumon carbon were added to a solution of (N-Cbz-Glu(O-t-Bu)-NHCH₂CH₂OCH₂)₂in 12 mL of MeOH and the resulting suspension was stirred under N₂ atroom temperature overnight. The mixture was diluted with CH₂Cl₂ andfiltered through a Celite pad. The solids were washed thoroughly withCH₂Cl₂ and the combined organic filtrates were concentrated to dryness.The residue was partitioned between CH₂Cl₂ and saturated aqueous NaHCO₃,washed with brine, dried over Na₂SO₄, and concentrated to give 1.13 g of(Glu(O-t-Bu)-NHCH₂CH₂OCH₂)₂ as a colorless oil; 1.44 (s, 18H), 1.81 (m,2H), 2.08 (m, 2H), 2.35 (m, 4H), 3.39 (dd, 2H, J=5, 7.5 Hz), 3.47 (t,4H, J=5 Hz), 3.58 (t, 4H, J=5 Hz), 7.53 (m, 2H).

Example 15 [N,N-bis(4-Benzyloxybenzyl)Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂ (cpd245)

[0123] A solution of 199 mg (0.384 mmol) of [Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂,403 mg (1.73 mmol) of 4-benzyloxybenzyl chloride, 30 mg (0.2 mmol) ofNaI, and 334 L (248 mg, 1.92 mmol) of DIEA was stirred under N₂ at roomtemperature for several days. The solution was partitioned between EtOAcand water and the organic layer was washed three times with water andonce with brine. After drying over Na₂SO₄, the solution was concentratedto give 528 mg of yellow oil which was purified by MPLC using a solventgradient ranging from 42-50% EtOAc/hexanes to afford 318 mg (64%) of cpd245 as a white foam; ¹H NMR (CDCl₃, 300 MHz) 1.42 (s, 18H), 2.01 (m,4H), 2.38 (m, 2H), 2.55 (m, 2H), 3.03 (dd, 2H, J=5, 8 Hz), 3.31 (m, 2H),3.4-3.6 (complex, 18H), 4.99 (s, 8H), 6.89 (d, 8H, J=8.5), 7.1-7.4(complex, 30H).

Example 16 [N,N-bis(4-Benzyloxybenzyl)GluNHCH₂CH₂OCH₂]₂ (cpd 246)

[0124] A solution of 219 mg (0.168 mmol) of cpd 245 in 2 mL of 33%TFA/CH₂Cl₂ was stirred ad room temperature overnight. The mixture wasconcentrated to give a crude product which was dissolved in HOAc andfreeze-dried to afford 251 mg of cpd 246 as an amber oil; ¹H NMR (CD₃OD,300 MHz) 2.1-2.6 (complex, 8H), 3.3-3.6 (complex, 8H), 3.57 (s, 4H),3.78 (m, 2H), 4.25 (broad d, 4H, J=14 Hz), 4.36 (broad d, 4H, J=14 Hz),5.09 (s, 8H), 7.03 (d, 8H, J=8 Hz), 7.3-7.5 (complex, 28H); MS (ES+) m/z1192 (MH+), 995, 596, 197 (base).

Example 17 [N-(3-Phenoxybenzyl)Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂

[0125] A solution of 680 mg (0.76 mmol) of [Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂and 278 μL (317 mg, 1.6 mmol) of 3-phenoxybenzaldehyde in 3 mL of TMOFwas stirred overnight at room temperature under N₂. The mixture wasconcentrated and pumped at high vacuum to give a colorless oil which wasdissolved in 3 mL of CH₂Cl₂ and treated with 678 mg (3.2 mmol) ofNaBH(OAc)₃. After stirring under N₂ for 2 days, 50 mL of saturatedaqueous NaHCO₃ was added and the mixture was extracted with CH₂Cl₂. Theorganic layers were combined, dried over Na₂SO₄, and concentrated andthe crude product (1.01 g) was purified by MPLC using a solvent gradientranging from 2-4% MeOH/CH₂Cl₂ to afford 490 mg of[N-(3-phenoxybenzyl)Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂ as a colorless oil; ¹HNMR (CDCl₃, 300 MHz) 1.41 (s, 18H), 1.89 (m, 4H), 2.31 (m, 4H), 3.12 (t,2H, J=6 Hz), 3.45 (m, 8H), 3.55 (s, 4H), 3.60 (d, 2H, J=13.5 Hz), 3.73(d, 2H, J=13.5 Hz), 6.86 (dd, 2H, J=1.5, 8 Hz), 7.00 (m, 8H), 7.2-7.4(complex, 8H); MS (ES+) m/z 883 (MH+), 589, 442, 414, 386 (base), 183.

Example 18[N-(3-Nitrobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂

[0126] DIEA (269 μL, 199 mg, 1.54 mmol), 3-nitrobenzyl bromide (322 mg,1.49 mmol), and [N-(3-phenoxybenzyl)Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂ (482 mg,0.546 mmol) were combined in 2 mL of DMF and heated at 60-70° C. underN₂ for 2 days. The reaction mixture was cooled and partitioned between100 mL of EtOAc and water. The organic layer was washed with three timeswith water and once with brine, dried over Na₂SO₄, and concentrated togive 661 mg (˜100%) of[N-(3-nitrobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂ whichwas used without purification; MS (ES+) m/z 1154 (MH+), 577, 130 (base).

Example 19[N-(3-Aminobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂

[0127] A solution of 661 mg (0.54 mmol) of crude[N-(3-nitrobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂ and2.71 g (12.0 mmol) of SnCl₂.2 H₂O in 20 mL of absolute EtOH was refluxedunder N₂ for 30 min. The cooled solution was poured into 500 mL of 2.5%aqueous Na₂CO₃ with rapid stirring and the resulting cloudy mixture wasextracted thoroughly with EtOAc. The slightly cloudy organic extractswere washed twice with brine, dried over Na₂SO₄, anc concentrated togive 604 mg of yellow oil which was purified by MPLC using 3%MeOH/CH₂Cl₂ to provide 350 mg (59%) of[N-(3-aminobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂ as apale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.41 (s, 18H), 1.97 (m, 4H),2.25 (m, 4H), 2.48 (m, 4H), 3.03 (dd, 2H, J=5, 8 Hz), 3.30 (m, 2H),3.4-3.8 (complex, 24H), 6.47 (d, 2H, J=7.5 Hz), 6.65 (m, 4H), 6.85 (d,2H, J=9.5 Hz), 6.9-7.15 (complex, 12H), 7.2-7.4 (complex, 8H); MS (ES+)m/z 1094 (MH+), 547 (base).

Example 20[N-(3-Phenoxybenzyl)-N-(3-(pentanoylamino)benzyl)-Glu-NHCH₂CH₂OCH₂]₂(cpd 247)

[0128] Pentanoyl chloride (16 uL, 16 mg, 0.136 mmol) was added dropwiseto a solution of 68 mg (0.062 mmol) of[N-(3-aminobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂, 20 μL(20 mg, 0.25 mmol) of pyridine in 0.3 mL of 1,2-dichloroethane. Themixture was shaken under N₂ overnight and was then partitioned betweenEtOAc and water. The organic layer was washed with saturated aqueousNaHCO₃, dried over Na₂SO₄, and concentrated to give 77 mg of[N-(3-phenoxybenzyl)-N-(3-(pentanoylamino)benzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂;MS (ES+) m/z 1073, 575 (base, MH+/2). The crude product was dissolved in1 mL of 50% TFA/CH₂Cl₂ and allows to stand overnight. The solution wasconcentrated and the resulting oil was dissolved in HOAc andfreeze-dried to provide 82 mg of cpd 247; ¹H NMR (CD₃OD, 300 MHz) 3.98(t, 6H, J=7.5 Hz), 1.39 (sextet, 4H, J=7.5 Hz), 1.66 (quintet, 4H, J=7.5Hz), 1.65 (m, 2H), 1.78 (m, 2H), 2.35 (t, 4H, J=7.5 Hz), 2.45 (m, 4H),3.38 (m, 4H), 3.50 (t, 2H, J=5), 3.57 (m, 4H), 4.10 (broad s, 8H),6.9-7.25 (complex, 14H), 7.25-7.4 (complex, 10H), 7.71 (s, 2H); MS (ES+)m/z 1150 (MH+), 575 (base).

Example 21 [N-Cbz-Lys(Boc)-NHCH₂CH₂]₃N

[0129] A solution of 1.0 g (2.63 mmol) of N-Cbz-Lys(Boc)OH, 0.131 mL(0.128 g, 0.876 mmol) of tris(2-aminoethyl)amine, 0.391 g (2.98 mmol) ofHOBt, 0.555 g (2.89 mmol) of EDCI, and 0.55 mL (0.408 g, 3.16 mmol) ofDIEA in 5 mL of CH₂Cl₂ was stirred under N₂ at room temperatureovernight. The mixture was diluted with EtOAc and washed with 10%aqueous citric acid, saturated aqueous NaHCO₃, and brine. The solutionwas dried over Na₂SO₄ and concentrated to give 0.872 g of[N-Cbz-Lys(Boc)-NHCH₂CH₂]₃N as an off-white solid; ¹H NMR (CD₃OD, 300MHz) 135 (m, 12H), 1.40 (s, 27H), 1.60 (m, 3H), 1.72 (m, 3H), 2.51 (m,6H), 2.99 (m, 6H), 3.10 (m, 3H), 3.21 (m, 3H), 4.12 (m, 3H), 5.00 (d,3H, J=12.5 Hz), 5.08 (d, 3H, J=12.5 Hz), 7.29 (m, 15H); MS (ES+) m/z1243 (base, MH+), 567, 467.

Example 22 [Lys(Boc)-NHCH₂CH₂]₃N

[0130] A solution of 0.841 g (0.682 mmol) [N-Cbz-Lys(Boc)-NHCH₂CH₂]₃N,0.252 g of 10% Pd—C, and 0.774 g (12.3 mmol) of ammonium formate in 10mL of MeOH was stirred for 5 h at room temperature under N₂. The mixturewas filtered through a Celite pad, the solids were washed with CH₂Cl₂,and the reslulting solution was concentrated to dryness. The residue waspartitioned between CH₂Cl₂ and brine; the organic layer was dried overNa₂SO₄ and concentrated to provide 0.191 g of [Lys(Boc)-NHCH₂CH₂]₃N asan off-white solid; ¹H NMR (CD₃OD, 300 MHz) 1.40 (s, 27H), 1.45 (m,12H), 1.75 (m, 6H), 2.62 (m, 6H), 3.01 (m, 6H), 3.28 (m, 6H), 3.64 (m,3H); MS (ES+) m/z 853 (MNa+), 831 (MH+), 266 (base).

Example 23 [N,N-bis(3-Phenoxybenzyl)Lys(Boc)-NHCH₂CH₂]₃N

[0131] A solution of 65 mg (0.078 mmol) of [Lys(Boc)-NHCH₂CH₂]₃N, 120 μL(140 mg, 0.70 mmol) of 3-phenoxybenzaldehyde, and 71 μL (65 mg, 0.70mmol) of borane-pyridine complexin 3 mL of absolute EtOH was stirred for4 days at room temperature under N₂. The mixture was concentrated todryness and partitioned between water and CH₂Cl₂. The organic layer wasconcentrated to give a yellow oil which was purified by MPLC using 2.5%MeOH/CH₂Cl₂ to give 78 mg of[N,N-bis(3-phenoxybenzyl)Lys(Boc)-NHCH₂CH₂]₃N as a yellow oil; MS (ES+)m/z 872 (base, [M-C₁₃H₁₂O)/2]+), 611, 443.

Example 24 [N,N-bis(3-Phenoxybenzyl)Lys-NHCH₂CH₂]₃N (cpd 277)

[0132] A solution of 78 mg (0.048 mmol) of[N,N-bis(3-phenoxybenzyl)Lys(Boc)-NHCH₂CH₂]₃N in 2 mL of 50% TFA/CH₂Cl₂was stirred for 2 h at room temperature. The mixture was diluted withCH₂Cl₂, washed with water and 5% Na₂CO₃, and concentrated to give 57 mgof cpd 277 as an off-white foam; ¹H NMR (CD₃OD, 300 MHz) 1.35 (m, 6H),1.52 (m, 6H), 1.76 (m, 6H), 2.75 (m, 6H), 3.19 (m, 6H), 3.40 (m, 6H),3.60 (m, 9H), 3.77 (m, 6H), 6.79 (d, 6H, J=8 Hz), 6.93 (m, 24H), 7.05(m, 6H), 7.19 (m, 6H), 7.29 (m, 12H); MS (ES+) m/z 813 ([MH₂/2]+), 721,542 (base, [MH/3]+).

Example 25 N,N-bis(3-Phenoxycinnamyl)Ser(t-Bu)-OMe (cpd 290) andN-(3-phenoxycinnamyl)Ser(t-Bu)-OMe (cpd 352)

[0133] A solution of 423 mg (2.0 mmol) of H-Ser(t-Bu)OMe.HCl, 1.01 g(3.5 mmol) of 3-phenoxycinnamyl bromide (Jackson, W. P.; Islip, P. J.;Kneen, G.; Pugh, A.; Wates, P. J. J.Med.Chem. 31 1988; 499-500), and0.87 mL (5.0 mmol, 650 mg) of DIEA in 6 mL of DMF was stirred under N₂at room temperature for 20 h. The mixture was partitioned between EtOAcand water and the organic layer was washed with water and brine. Afterdrying over Na₂SO₄, the organic solution was concentrated to give 0.98 gof yellow oil. The crude residue was purified by MPLC using a solventgradient ranging from 10-30% EtOAc/hexanes to give two products. Theless polar product (168 mg, 14% based on starting amino acid),N,N-bis(3-phenoxycinnamyl)Ser(t-Bu)-OMe (cpd 290), was isolated as apale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.15 (s, 9H), 3.35 (dd, 2H,J=7, 14.5 Hz), 3.53 (dd, 2H, J=5.5, 14.5 Hz), 3.6-3.8 (complex, 3H),3.69 (s, 3H), 6.18 (dt, 2H, J=16, 6.5 Hz), 6.49 (d, 2H, J=16 Hz), 6.86(dd, 2H, J=2, 8 Hz), 6.9-7.4 (complex, 16H); MS (ES+) m/z 614, 592 (MH+,base), 406, 384, 209.

[0134] The more polar product (354 mg, 46% based on starting aminoacid), N-(3-phenoxycinnamyl)Ser(t-Bu)-OMe (cpd 352), was isolated as apale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.15 (s, 9H), 1.98 (broad s,1H), 3.32 (ddd, 1H, J=1.2, 6.5, 14 Hz), 3.4-3.7 (complex, 4H), 3.72 (s,3H), 6.21 (dt, 1H, J=16, 6.5 Hz), 6.48 (d, 1H, J=16 Hz), 6.88 (dd, 1H,J=1.5, 8 Hz), 7.0-7.4 (complex, 8H); MS (ES+) m/z 789 (2M+Na+), 384(MH+, base), 209.

Example 26 N,N-Bis(3-phenoxycinnamyl)Ser-OMe (cpd 299)

[0135] N,N-Bis(3-phenoxycinnamyl)Ser(t-Bu)-OMe (cpd 290, 168 mg, 0.284mmol) was stirred in 3 mL of 50% TFA/CH₂Cl₂ under N₂ overnight. Thesolvent was removed using a rotary evaporator and the crude residue waspartitioned between EtOAc and saturated aqueous NaHCO₃. After washingwith brine and drying over Na₂SO₄, the organic layer was concentratedusing a rotary evaporator and the crude product (134 mg) was purified byMPLC using 30% EtOAc/hexanes to give 44 mg (29%) ofN,N-bis(3-phenoxycinnamyl)Ser-OMe (cpd 299) as a colorless oil; ¹H NMR(CDCl₃, 300 MHz) 1.6 (broad s, 2H), 3.38 (dd, 2H, J=8, 12 Hz), 3.4-3.9(complex, 5H), 3.72 (s, 3H), 6.13 (dt, 2H, J=16, 7 Hz), 6.50 (d, 2H,J=16 Hz), 6.8-7.4 (complex, 18H); MS (ES+) m/z 536 (MH+).

Example 27 N,N-Bis(3-phenoxycinnamyl)Ser-OH (cpd 300)

[0136] N,N-Bis(3-phenoxycinnamyl)Ser-OMe (cpd 299, 44 mg, 0.082 mmol)was dissolved in 0.2 mL of MeOH and was stirred with 0.090 mL of 1Naqueous NaOH. When TLC analysis revealed that starting material had beenconsumed, the solvent was removed by rotary evaporation and the residuewas lyophilized from acetic acid to give 42 mg (88%) ofN,N-bis(3-phenoxycinnamyl)Ser-OH acetate (cpd 300) as a sticky yellowsolid; ¹H NMR (methanol-d₄, 300 MHz) 1.97 (s, 3H), 3.3-4.2 (complex,7H), 6.80 (d, 2H, J=16 Hz), 6.9-7.4 (complex, 18H); MS (ES+) m/z 522(MH+), 209.

Example 28 N-(3-Phenoxycinnamyl)Ser-OMe (cpd 346)

[0137] N-(3-Phenoxycinnamyl)Ser(t-Bu)-OMe (cpd 352, 268 mg, 0.699 mmol)was stirred in 3 mL of 50% TFA/CH₂Cl₂ under N₂ overnight. The solventwas removed using a rotary evaporator and the crude residue (256 mg) waspurified by MPLC using EtOAc to give 137 mg (60%) ofN-(3-phenoxycinnamyl)Ser-OMe (cpd 346) as a colorless oil; ¹H NMR(CDCl₃, 300 MHz) 2.2 (broad s, 2H), 3.36 (dd, 1H, J=6, 14 Hz), 3.4-3.5(complex, 2H), 3.62 (dd, 1H, J=6.5, 11 Hz), 3.74 (s, 3H), 3.80 (dd, 1H,J=4.5, 11 Hz). 6.19 (dt, 1H, J=16, 6.5 Hz), 6.48 (d, 1H, J=6 Hz), 6.88(dd, 1H, J=1.5, 8 Hz), 7.0-7.4 (complex, 8H); MS (ES+) m/z 677 (2M+Na+),350 (M+Na+), 328 (MH+), 209 (base).

Example 29 N-(3-phenoxycinnamyl)Ser-OH (cpd 347)

[0138] N-(3-Phenoxycinnamyl)Ser-OMe (cpd 346, 110 mg, 0.336 mmol) wasdissolved in 1.5 mL of MeOH and was stirred with 0.50 mL of 1N aqueousNaOH. When TLC analysis revealed that starting material had beenconsumed, the solvent was removed by rotary evaporation. The residue wasdissolved in water and acidified to pH 7-8 with 1N aqueous HCl; theresulting solids were filtered, washed with water, and dried to give 71mg of white powder. The insoluble powder was dissolved in TFA and, afterremoval of excess TFA by rotary evaporation, lyophilized from aceticacid to give 82 mg (57%) of N-(3-phenoxycinnamyl)Ser-OH trifluoroacetate(cpd 347) as an amber oil; ¹H NMR (methanol-d₄, 300 MHz) 3.88 (d, 2H,J=7H), 4.0-4.2 (complex, 3H), 6.27 (dt, 1H, J=16, 6.5), 6.83 (d, 1H,J=16 Hz), 6.9-7.4 (complex, 9H); MS (ES+) m/z 314, (MH+), 209.

Example 30 N-(3-Phenoxycinnamyl)Glu(O-t-Bu)-OH (cpd 337)

[0139] A mixture of 249 mg (0.585 mmol) ofN-(3-phenoxycinnamyl)Glu(O-t-Bu)-OMe (cpd 334) in 3 mL of MeOH wassonicated to speed dissolution, and the resulting solution was treatedwith 0.585 mL of 1N aqueous NaOH. After stirring overnight, the MeOH wasremoved using a rotary evaporator and the residue was dissolved inwater. Acidification with 0.64 mL of 1N aqueous HCl produced a 250 mg ofsolid material that was triturated with Et₂O to give 111 mg (46%) ofN-(3-phenoxycinnamyl)Glu(O-t-Bu)-OH (cpd 337) as a white solid; ¹H NMR(300 MHz, methanol-d₄) 1.43 (s, 9H), 1.9-2.2 (complex, 2H), 2.46 (t, 2H,J=7 Hz), 3.57 (dd, 1H, J=5, 7 Hz), 3.78 (dd, 1H, J=7, 13.5 Hz), 3.82(dd, 1H, J=7, 13.5 Hz), 6.28 (dt, 1H, J=16, 7 Hz), 6.81 (d, 1H, J=16Hz), 6.9-7.5 (complex, 9H); MS (ES+) m/z 412 (MH+, base), 356, 209.Anal. Calcd for C₂₄H₂₉NO₅.0.4 H2O: C, 68.55; H, 7.04; N, 3.24. Found: C,68.89; H, 7.04; N, 3.24.

Example 31 N-(3-Phenoxycinnamyl)Glu-OH (cpd 326)

[0140] A mixture of 85 mg (0.21 mmol) ofN-(3-phenoxycinnamyl)Glu(O-t-Bu)-OH (cpd 337) in was stirred in 1 mL of50% TFA/CH₂Cl₂ for 1 h. After solvent removal using a rotary evaporator,the residue was dissolved in acetic acid and freeze-dried to give 75 mg(76%) of N-(3-phenoxycinnamyl)Glu-OH tifluoroacetate (cpd 326) as afluffy white solid; ¹H NMR (300 MHz, methanol-d₄) 2.0-2.4 (complex, 2H),2.55 (m, 2H), 3.84 (d, 2H, J=7 Hz), 3.96 (dd, 1H, J=5, 7 Hz, 6.24 (dt,1H, J=16, 7 Hz), 6.84 (d, 1H, J=16 Hz), 6.9-7.4 (complex, 9H); MS (ES+)m/z 356 (MH+), 209 (base). TABLE 2

R¹ (amino cpd % inh acid side chain) R² R³ W, Q 11 70 Asn, Asp, Gln, Glu3-PhO CH═CH 12 59 Cys, Met, Ser, Thr 3-PhO CH═CH 13 nd Arg, Gly, His,Pro 3-PhO CH═CH 14 30 Lys(2-Cl-Cbz), 3-PhO CH═CH Phe, Trp, Tyr 15 48Ala, Ile, Leu, Val 3-PhO CH═CH 16 nd Glu, Asp 2,3-benzo CH═CH 17 nd Cys,Met 2,3-benzo CH═CH 18 nd Ser, Thr 2,3-benzo CH═CH 19 nd His, Arg(Mtr)2,3-benzo CH═CH 20 nd Pro, Gly 2,3-benzo CH═CH 21 nd Phe, Tyr 2,3-benzoCH═CH 22 nd Trp, Lys(2-Cl-Cbz) 2,3-benzo CH═CH 23 nd Ile, Ala 2,3-benzoCH═CH 24 nd Val, Leu 2,3-benzo CH═CH 25 nd Asn, Lys 2,3-benzo CH═CH 26nd Ala, Ile 3,4-benzo CH═CH 27 nd Arg(Mtr), 3,4-benzo CH═CHLys(2-Cl-Cbz) 28 nd Asp, Glu 3,4-benzo CH═CH 29 nd Cys, Met 3,4-benzoCH═CH 30 nd Gly, Pro 3,4-benzo CH═CH 31 nd His, Lys 3,4-benzo CH═CH 32nd Leu, Val 3,4-benzo CH═CH 33 nd Lys(2-Cl-Cbz), Phe 3,4-benzo CH═CH 34nd Ser,Thr 3,4-benzo CH═CH 35 nd Trp,Tyr 3,4-benzo CH═CH

[0141] TABLE 3

EPO/EBP-Ig cpd % inh @ 50 μM R¹ R² R⁹ W,Q MS MH+ 36 nd CH₃ 4-CF₃ H CH═CH458 37 19 H 4-CF₃ H CH═CH 430 38 nd (CH₂)₄NH(2-Cl-Cbz) 4-F H CH═CH 44840 nd CH₃ 4-F H CH═CH 223 41 nd CH₂CO₂H 4-F H CH═CH 266 42 nd CH₂CH₂CO₂H4-F H CH═CH 281 43 nd (CH₂)₃NHC(═NH)NH₂ 4-F H CH═CH 308 45 nd PhCH₂ 4-FH CH═CH 299 46 nd 4-HO-PhCH₂ 4-F H CH═CH 315 47 nd CH₂OH 4-F H CH═CH 23848 nd CH(OH)CH₃ 4-F H CH═CH 253 49 1 (CH₂)₃NHC(═NH)NH₂ H H S 419 50 −6(CH₂)₄NH₂ H H S 391 51 nd CH(CH₃)CH₂CH₃ H H S 376 52 21 CH₂CH₂CO₂H H H S392 53 14 CH₂CO₂H H H S 378 54 18 CH₃ H H S 334 55 4 CH₂CH₂CONH₂ H H S391 56 nd (CH₂)₄NHCbz H Me S 539 57 0 (CH₂)₄NHCbz H CH₂Ph S 615 58 ndCH₂(indol-3-yl) H Me S 463 59 26 CH₂CH₂CO₂-t-Bu H Me S 462 60 9CH₂CH₂CO₂Et H Me S 434 61 14 CH₂CH₂CO₂H H Me S 406

[0142] TABLE 4

EPO/EBP-Ig cpd % inh @ 5 μM R^(a) R² R⁴ R⁹ MS,MH+ 62 nd t-Bu NO₂ NO₂t-Bu 516 63 20 H PhCH₂NH PhO H 511 64 −4 H 4-MePhCONH 4-MePhCONH H 58065 −7 H 4-MePhSO₂NH 4-MePhSO₂NH H 652 66 −16 H 3-ClPhCH₂NH PhO H 546 67−8 H 3-BrPhCH₂NH PhO H 590 68 −13 H 2-FPhCH₂NH PhO H 529 69 −13 H2-MePhCH₂NH PhO H 525 70 −8 H 4-FPhCH₂NH PhO H 529 71 −6 H 3-ClPhCH₂NH4-Me-PhO H 560 72 −14 H F₅-PhCH₂NH 4-Me-PhO H 615 73 −13 H 2-FPhCH₂NH4-Me-PhO H 543 74 −7 H 3-CNPhCH₂NH 4-Me-PhO H 550 75 −2 H PhCH₂NH4-Me-PhO H 525

[0143] TABLE 5

EPO/EBP-Ig MS, cpd % inh @ 50 μM R^(a) R² R³ R⁴ R⁵ R⁹ n MH+ 76 25 t-BuPhO H PhO H t-Bu 1 636 77 52 H PhO H PhO H H 1 524 78 nd H H 4-MePhCONHH BnO H 2 593 79 nd H H n-BuCONH H BnO H 2 559 80 nd H H 2-naphthyl CONHH BnO H 2 629 81 nd H H 2-furyl CONH H BnO H 2 569 82 32 H H4-MeO-PhCONH H BnO H 2 609 83 18 H H HO₂C(CH₂)₃CONH H BnO H 2 589 84 14H H C₂F₅CONH H BnO H 2 621 85 20 H H CF₃CONH H BnO H 2 571 86 37 H H4-pyridyl-CONH H BnO H 2 580 87 23 H H 4-MePhSO₂NH H BnO H 2 629 88 10.3H H HO₂CCH₂(1,1- H BnO H 2 643 cyclopentyl) CH₂CONH 89 22 H H PhOCONH HBnO H 2 595 90 29 H H 4-Ph-PhCONH H BnO H 2 655 91 19 H H 4-NO₂-PhCONH HBnO H 2 624

[0144] TABLE 6

EPO/EBP-Ig MS, cpd % inh @ 50 μM R^(a) R² R³ R⁴ R⁵ R⁶ R⁹ MH+  92 20 H HH H H 2 Me 394  93 20 t-Bu H H H H 2 Me 450  94 25 Et H H H H 2 Me 422 95 15 t-Bu 2,3-benzo 2,3-benzo 2 Me 550  96 −5 t-Bu PhO H PhO H 2 Me634  97 14 t-Bu 3,4-benzo 3,4-benzo 2 H 536  98 12 t-Bu H Ph H Ph 2 Me602  99 13 t-Bu 3,4-di-Cl-PhO H 3,4-di-Cl-PhO H 2 Me 772 100 34 H H Ph HPh 2 Me 546 101 32 H 3,4-di-Cl-PhO H 3,4-di-Cl-PhO H 2 Me 716 102  5t-Bu 4-t-Bu-PhO H 4-t-Bu-PhO H 2 t-Bu 789 103 17 t-Bu 3-CF3-PhO H3-CF3-PhO H 2 t-Bu 812 104 78 H 4-t-Bu-PhO H 4-t-Bu-PhO H 2 H 676 105 70H 3-CF3-PhO H 3-CF3-PhO H 2 H 700 106 20 t-Bu PhO H PhO H 1 t-Bu 662 10778 H PhO H PhO H 2 H  562* 108 81 H PhO H PhO H 1 H 550

[0145] TABLE 7

EPO/ EBP-Ig % inh MS, @ 50 MH cpd μM R^(a) R² R³ R⁴ R⁵ R⁹ + 109  7 BocBnO H BnO H Me 653 110 54 H H BnO H BnO Me 553 111  5 Boc H BnO H BnO Me653 112 59 H BnO H BnO H Me 553 113 24 Boc H BnO NO₂ H Me 592 114 37 H HBnO NO₂ H Me 492 115 35 H H BnO NH₂ H Me 462 116 32 H H BnO n-BuCONH HMe 546 117 34 H H BnO 2-furylCONH H Me 556 118 36 H H BnO 4-MePhCONH HMe 580 119 34 H H BnO i-Pr-CONH H Me 532 120 35 H H BnO 4-pyridyl- H Me567 CONH 121 45 H H BnO 2-naphthyl- H Me 616 CONH 122 nd Boc PhCH₂NH HPhCH₂NH H Me 651 123 nd Boc 2-MePhCH₂NH H 2-MePhCH₂NH H Me 679 124 ndBoc 4-MeO- H 4-MeO- H Me 711 PhCH₂NH PhCH₂NH 125 nd Boc 3,4-di-MeO H 3,4di-MeO H Me 771 PhCH₂NH PhCH₂NH 126 nd Boc —NH₂ H —NH₂ H Me 471 127 nd HPhCH₂NH H PhCH₂NH H Me 551 128 nd H 2-MePhCH₂NH H 2-MePhCH₂NH H Me 579129 nd H 4-MeO H 4-MeO H Me 611 PhCH₂NH PhCH₂NH 130 nd H 3,4-di-MeO- H3,4-di-MeO- H Me 671 PhCH₂NH PhCH₂NH 131 nd H PhCH₂NH H PhCH₂NH H Me 579132 nd HO₂CCH₂ PhCH₂CH₂NH H PhCH₂CH₂NH H Me 651 CH₂CO 133 nd HO₂CCH₂2-MePhCH₂NH H 2-MePhCH₂NH H Me 679 CH₂CO 134 nd HO₂CCH₂ 4-MeO- H 4-MeO-H Me 711 CH₂CO PhCH₂NH PhCH₂NH 135 nd HO₂CCH 3,4-di-MeO- H 3,4-di-MeO- HMe 771 CH₂CO PhCH₂NH PhCH₂NH 136 nd HO₂CCH₂ PhCH₂CH₂NH H PhCH₂CH₂NH H Me679 CH₂CO

[0146] TABLE 8

EPO/EBP-Ig % inh @ 50 MS, cpd μM R^(a) R² R⁴ R⁵ R⁹ MH+ 137 nd H PhO PhOH Me 551 138 nd Boc 4-t-Bu-PhO BnO H Me 721 139 nd H 4-t-Bu-PhO BnO H Me621 140 nd H (CF₃CO)₂N BnO H H 666 141 nd H PhCONH BnO H H 578 142 nd H4-pyridyl-CONH BnO H H 579 143 nd H (CF₃CO)₂N PhO H H 652 144 nd HPhCONH PhO H H 564 145 nd H 4-pyridyl-CONH PhO H H 565 146 nd H(CF₃CO)₂N MeO MeO H 620 147 nd H PhCONH MeO MeO H 532 148 nd H4-pyridyl-CONH MeO MeO H 533 149 nd H (CF₃CO)₂N H PhO H 652 150 nd HPhCONH H PhO H 564 151 nd H 4-pyridyl-CONH H PhO H 565 152 nd H PhCONH HBnO H 578 153 nd H 4-pyridyl-CONH H BnO H 579 154 nd H (CF₃CO)₂N H BnO H666 155 nd HO₂CCH₂CH₂CO 4-MeO-PhCONH PhO H H 694 156 nd HO₂CCH₂CH₂COPhCONH PhO H H 664 157 nd HO₂CCH₂CH₂CO 2-naphthyl- PhO H H 714 CONH 158nd HO₂CCH₂CH₂CO 4-Me-PhSO₂NH PhO H H 714 159 nd HO₂CCH₂CH₂CO4-MeO-PhCONH 2,3- H 652 benzo 160 nd HO₂CCH₂CH₂CO PhCONH 2,3- H 622benzo 161 nd HO₂CCH₂CH₂CO 2-naphthyl- 2,3- H 672 CONH benzo 162 ndHO₂CCH₂CH₂CO 4-Me-PhSO₂NH 2,3- H 672 benzo 163 nd HO₂CCH₂CH₂CO4-MeO-PhCONH H F H 620 164 nd HO₂CCH₂CH₂CO PhCONH H F H 590 165 ndHO₂CCH₂CH₂CO 2-naphthyl- H F H 640 CONH 166 nd HO₂CCH₂CH₂CO 4-Me-PhSO₂NHH F H 640 167 nd HO₂CCH₂CH₂CO 4-MeO-PhCONH BnO H H 708 168 ndHO₂CCH₂CH₂CO PhCONH BnO H H 678 169 nd HO₂CCH₂CH₂CO 2-naphthyl- BnO H H728 CONH 170 nd HO₂CCH₂CH₂CO 4-Me-PhSO₂NH BnO H H 728

[0147] TABLE 9

EPO/ EBP- Ig % inh MS, @ 50 MH cpd μM R^(a) R² R³ R⁴ R⁵ R⁹ + 171 nb CbzH H H H Me 527 172 15 Cbz H H H H H 513 173  5 Cbz H H H H t- 569 Bu 17423 Cbz H MeO H MeO Me 587 175  1 Cbz 3,4- 3,4- Me 627 benzo benzo 176 −4Cbz PhO H PhO H Me 711 177 nd Cbz 2,3-benzo 2,3-benzo Me 627 178 36 BocH NO₂ H NO₂ Me 583 179 30 Boc H NO₂ H NO₂ H 569 180 −4 Boc PhO H PhO HMe 677 181 −9 Boc 4-t-Bu-PhO H 4-t-Bu-PhO H Me 790 182 18 H 4-t-Bu-PhO H4-t-Bu-PhO H Me 689 183 36 Boc NO₂ H NO₂ H Me 583 184 53 H NO₂ H NO₂ HMe 483 185 29 H NH₂ H NH₂ H Me 423 186 nd H n-Bu-CONH H n-Bu-CONH H Me591 187 nd H 2-furyl-CONH H 2-furyl-CONH H Me 611 188 nd H PhCONH HPhCONH H Me 631 189 nd H 4-Me-PhCONH H 4-Me-PhCONH H Me 659 190 nd H4-NO₂-PhCONH H 4-NO₂-CONH H Me 721 191 nd H 4-Me-PhSO₂NH H4-Me-PhSO₂CONH H Me 731 192 nd H Cbz-NH H Cbz-NH H Me 691 193 nd H4-Br-PhCONH H 4-Br-PhCONH H Me 789 194 nd H 2-MeO-PhCONH H 2-MeO-PhCONHH Me 691 195 nd H 3-MeO-PhCONH H 3-MeO-PhCONH H Me 691 196 nd H4-MeO-PhCONH H 4-MeO-PhCONH H Me 691 197 nd H CH₃CH═CHCON H CH₃CH═CHCONH Me 559 H H 198 nd H C₂F₅CONH H C₂F₅CONH H Me 715 199 nd H 2-naphthyl-H 2-naphthyl- H Me 731 CONH CONH 200 nd H EtO₂CCH₂CH₂C H EtO₂CCH₂CH₂CO HMe 679 ONH NH 201 nd H CF₃CONH H CF₃CONH H Me 615 202 nd H MeSO₂NH HMeSO₂NH H Me 579

[0148] TABLE 10

EPO/ EBP-Ig MS, % inh @ MH cpd 50 μM R^(a) R² R³ R⁴ R⁵ Z + 203 37 Boc HH H H 4-(MeCOCH₂CH₂)-PhNH 640 204 −6 H H H H H 4-(MeCOCH₂CH₂)-PhNH 540205 26 H H H H H n-Bu-NH 434 206 17 2-MeO-PhCO H H H H n-Bu-NH 568 20720 4-MeO-PhCO H H H H n-Bu-NH 568 208 22 PhCO H H H H n-Bu-NH 538 209 252-MeO-PhCO H H H H n-Bu-NH 568 210 nd Boc H H H H 4-MeO-PhCH₂CH₂NH 612211 62 H H H H H 4-MeO-PhCH₂CH₂NH 512 212 −10 H H H H H n-Pr-NH 420 214nd Boc H H H H 3,4-di-MeO- 642 PhCHCHNH 215 nd Boc H H H H3-MeO-PhCH₂CH₂NH 612 216 10 Boc H H H H 4-(PhCH═CHCH₂O)— 700 PhCH₂NH 217nd Boc H H H H 4-HO-PhCH₂NH 584 218 nd Boc H H H H EtNH 506 219 nd Boc HH H H MeNH 492 220 45 H H H H H 4-(PhCH═CHCH₂O)- 600 PhCH₂NH 221 48 H HH H H 3,4-di-MeO- 542 PhCH₂CH₂NH 222 56 H H H H H 3-MeO-PhCH₂CH₂NH 512223 nd Boc H H H H 2-MeO-PhCH₂CH₂NH 612 224 51 H H H H H2-MeO-PhCH₂CH₂NH 512 225 10 Boc PhO H PhO H 4-MeO-PhCH₂CH₂NH 797 226 ndBoc H H H H PhCH₂CH₂NH 582 227 48 H H H H H PhCH₂CH₂NH 482 228 21 PhNHCOPhO H PhO H 4-MeO-PhCH₂CH₂NH 816 229 22 4-PhO- H H H H 4-MeO-PhCH₂CH₂NH723 PhNHCO 230 42 3,4-di-Cl- H H H H 4-MeO-PhCH₂CH₂NH 700 PhNHCO 231 364-EtO2C- H H H H 4-MeO-PhCH₂CH₂NH 703 PhNHCO 232 14 4-PhO- PhO H PhO H4-MeO-PhCH₂CH₂NH 908 PhNHCO 233 18 H H NO H NO 3-MeO-PhCH₂CH₂NH 602 2 2234 nd Boc H H H H PhCH₂NH 568 235 49 H H H H H PhCH₂NH 468 236 nd Boc HPh H Ph 4-MeO-PhCH₂CH₂NH 765 237 55 HO₂CCH₂CH₂C H H H H 3-MeO-PhCH₂CH₂NH612 O 238 39 H H Ph H Ph 4-MeO-PhCH₂CH₂NH 664 239 46 H PhO H PhO HPhCH₂CH₂NH 666 240 nd HO₂CCH₂CH₂C PhO H PhO H PhCH₂CH₂NH 780 H₂CO 285 40H H H H H 4-(NH₂CO)piperidin-1-yl 489

[0149] TABLE 11

EPO/ EBP-Ig % inh @ MS, cpd 50 μM R^(a) R² R³ R⁴ R⁵ Z r [MH₂/2]+ 241 2t-Bu H BnO H BnO NH(CH₂)₃O(CH₂)₄O 1 666 (CH₂)₃NH 242 1 t-Bu H BnO H BnONH(CH₂)₃O(CH₂CH 1 674 ₂O)₂(CH₂)₃NH 243 75 H H BnO H BnO NH(CH₂)₃O(CH₂)₄O1 610 (CH₂)₃NH 244 66 H H BnO H BnO NH(CH₂)₃O(CH₂CH 1 618 ₂O)₂(CH₂)₃NH245 0 t-Bu H BnO H BnO NH(CH₂)₂O(CH₂)₂O 2 652 (CH₂)₂NH 246 79 H H BnO HBnO NH(CH₂)₂O(CH₂)₂O 2 596 (CH₂)₂NH 247 47 H n-Bu- H PhO HNH(CH₂)₂O(CH₂)₂O 2 575 CONH CH₂)₂NH 248 56 H 2-furyl- H PhO HNH(CH₂)₂O(CH₂)₂O 2 585 CONH (CH₂)₂NH 249 72 H 4-Me- H PhO HNH(CH₂)₂O(CH₂)₂O 2 609 PhCON (CH₂)₂NH H 250 78 H 4-Me- H PhO HNH(CH₂)₂O(CH₂)₂O 2 645 PhSO₂N (CH₂)₂NH H

[0150] TABLE 12

EPO/EBP-Ig % inh @ 50 MS cpd μM R^(a) R² R⁴ Z r [MH₂/2]+ 251 49 H H HNH(CH₂)₂O(CH₂)₂ 2 436 O(CH₂)₂NH 252 −4 t-Bu 4-t-Bu- 4-t-Bu-PhONH(CH₂)₃O(CH₂)₄ 1 803 PhO O(CH₂)₃NH 253 −5 t-Bu 4-t-Bu- 4-t-BuPhONH(CH₂)₃O(CH₂C 1 811 PhO H₂O₂CH₂)₃NH 254 −9 t-Bu 4-t-Bu- 4-t-Bu-PhONH(CH₂)₁₀NH 1 787 PhO 255 0 t-Bu PhO 4-t-Bu-PhO NH(CH₂)₁₂NH 1 801 256 10t-Bu 4-t-Bu- 4-t-Bu-PhO NH(CH₂)₂O(CH₂)₂ 1 789 PhO O(CH₂)₂NH

[0151] TABLE 13

EPO/EBP-Ig MS, cpd % inh @ 50 μM R^(a) Z [MH₂/2]+ 257 −26 BocNH(CH₂)₃O(CH₂CH₂O)₂ 731 (CH₂)₃NH 258 −24 Boc NH(CH₂)₃O(CH₂)₄O(CH 723₂)₃NH 259 −13 Boc NH(CH₂)₁₂NH 721 260 −12 Boc NH(CH₂)₂O(CH₂)₂O(CH 695₂)₂NH 261 51 H NH(CH₂)₂O(CH₂)₂O(CH 595 ₂)₂NH 262 93 HO₂CCH₂CH₂CONH(CH₂)₂O(CH₂)₂O(CH 695 ₂)₂NH 263 88 HO₂C(CH₂)₃CO NH(CH₂)₂O(CH₂)₂O(CH709 ₂)₂NH 264 89 HO₂CCH₂CMe₂CH₂CO NH(CH₂)₂O(CH₂)₂O(CH 737 ₂)₂NH 265 65HO₂CCH₂CH₂CO NH(CH₂)₃O(CH₂)₄O(CH 723 ₂)₃NH 266 82 HO₂C(CH₂)₃CONH(CH₂)₃O(CH₂)₄O(CH 737 ₂)₃NH 267 83 HO₂CCH₂CMe₂CH₂CONH(CH₂)₃O(CH₂)₄O(CH 765 ₂)₃NH 268 40 HO₂CCH₂CMe₂CH₂CO NH(CH₂)₁₂NH 764269 55 HO₂CCH₂CH₂CH₂CO NH(CH₂)₁₂NH 735 270 56 HO₂CCH₂CH₂CO NH(CH₂)₁₂NH721 271 77 HO₂CCH₂CH₂CO NH(CH₂)₃O(CH₂CH₂O)₂ 731 (CH₂)₃NH 272 78HO₂CCH₂CH₂CH₂CO NH(CH₂)₃O(CH₂CH₂O)₂ 745 (CH₂)₃NH

[0152] TABLE 14

EPO/ EBP-Ig MS, % inh @ [MH₂/2] cpd 50 μM R^(a) R² R⁴ Z n + 273 ndHO₂CCH₂C 4-Me-PhO 4-Me-PhO NH(CH₂)₂O(CH₂)₂O( 2 695 H₂CO CH₂)₂NH 274 ndHO₂CCH₂C PhO PhO NH(CH₂)₂O(CH₂)₂O( 2 667 H₂CO CH₂)₂NH 275 nd HO₂CCH₂C4-MeO- 4-MeO- NH(CH₂)₂O(CH₂)₂O( 2 727 H₂CO PhO PhO CH₂)₂NH 276 ndHO₂CCH₂C 4-t-Bu- 4-t-Bu- NH(CH₂)₂O(CH₂)₂O( 2 780 H₂CO PhO PhO CH₂)₂NH277 nd H PhO PhO NHCH₂CH₂)₃N 3 813 278 nd H 4-Me-PhO 4-Me-PhO(NHCH₂CH₂)₃N 3 855 279 nd H 4-Me- 4-MeO- (NHCH₂CH₂)₃N 3 903 PhO PhO 280nd HO₂CCH₂C 4-MeO- 4-MeO- (NHCH₂CH₂)₃N 3 1053  H₂CO PhO PhO 281 ndHO₂CCH₂C 4-Me-PhO 4-Me-PhO (NHCH₂CH₂)₃N 3 1005  H₂CO 282 nd HO₂CCH₂C PhOPhO HCH₂CH₂)₃N 3 963 H₂CO 283 nd Boc PhO PhO NH(CH₂)₃NMe 2 666 (CH₂)₃NH284 nd Boc 4-Me-PhO 4-Me-PhO NH(CH₂)₃NMe 2 694 (CH₂)₃NH

[0153] TABLE 15

EPO/EBP-Ig cpd % inh @ 50 μM R¹ R² R³ MS, MH+ 285 −28   Me H H 473 28646 H BnO H 565 287 36 H 4-Me-PhO H 565 288 27 H 4-tBu-PhO H 607 289 20 HH PhO 551

[0154] TABLE 16A

EPO/EBP-Ig cpd % inh @ 50 μM R¹ R² R³ R⁴ 290  0 Me PhO H t-Bu 291 17 MeH Ph t-Bu 292 11 Me 3-CF3-C6H4O H t-Bu 293 14 Me 3,4-Cl2-C6H3O H t-Bu294  9 Me 4-t-Bu-C6H4O H t-Bu 295 10 Me H Ph H 296  0 Me 3,4-Cl2-C6H3O HH 297  0 Me 3-CF3-C6H4O H H 298  1 Me 4-t-Bu-C6H4O H H 299 nd Me PhO H H300 57 H PhO H H 301 25 H H Ph t-Bu 302 30 H 3,4-Cl2-C6H3O H t-Bu 303 21H 3-CF3-C6H4O H t-Bu 304 19 H 4-t-Bu-C6H4O H t-Bu 305 48 H H Ph H 306 21Me H H t-Bu 307 25 H 3,4-Cl2-C6H3O H H 308 25 H 3-CF3-C6H4O H H 309 13 H4-t-Bu-C6H4O H H 310 34 Me H H H

[0155] TABLE 16B MS, cpd MPLC solvent appearance empirical formula MH+290 10-30% pale yellow oil C38H41NO5 592 EtOAc/hex 291 1:5 EtOAc/hexyellow oil C38H41NO3 560 292 1:5 EtOAc/hex yellow oil C40H39F6NO5 728293 1:5 EtOAc/hex yellow oil C38H37C14NO5 728 294 1:5 EtOAc/hex yellowoil C46H57NO5 704 295 off-white solid C34H33NO3/1 504 C2H4O2 296 ambersolid C34H29C14NO5/1 672 C2H4O2 297 amber oil C36H31F6NO5/1 672 C2H4O2298 off-white solid C42H49NO5/1 648 C2H4O2 299 30% EtOAc/hex colorlessoil C34H33NO5 536 300 sticky C33H31NO5/1 522 yellow solid C2H4O2 301yellow solid C37H39NO3 546 302 amber oil C37H35C14NO5 714 303 amber oilC39H37F6NO5 714 304 amber oil C45H55NO5 690 305 amber solid C33H31NO2/1490 C2HF3O2 306 light-yellow oil C26H33NO3/0.25 408 H2O 307 amber solidC33H27C14NO5/1 658 C2HF3O2 308 amber oil C35H29F6NO5/1 658 C2HF3O2 309off-white solid C41H47NO5/1 634 C2HF3O2 310 light yellow oil C22H25NO3/1352 C2H4O2

[0156] TABLE 17A

EPO/EBP-Ig cpd % inh @ 50 μM R¹ R² R³ 311 5.3 t-Bu PhO H 312 45 H PhO H

[0157] TABLE 17B cpd MPLC solvent appearance empirical formula MS, MH+311 10% EtOAc/hex pale yellow oil C36H37NO4 548 312 sticky brownC32H29NO4/1 492 solid C2HF3O2

[0158] TABLE 18A

EPO/EBP-Ig, cpd % inh @ 50 μM R¹ R² R³ R⁴ 313 28 H H CF3 (CH2)4NH(2Cl-Cbz 314 12 Me H CO2H (CH2)4NH2 315 nd Me H NO2 (CH2)4NHBoc 316 20 Me OPhH (CH2)4NHBoc 317 13 Me 4-t-Bu-C6H4O H (CH2)4NHBoc 318 14 Me H H(CH2)4NHBoc 319 nd Me H H (CH2)4NHCbz 320 17 Me H OMe (CH2)4NHCbz 321 42Me CO2Me H (CH2)4NHCbz 322 nd Me H 2,3-benzo (CH2)4NHCbz 323  6 Me HCO2H (CH2)4NHCbz 324 nd Me H CO2Me nd

[0159] TABLE 18B MS, cpd MPLC solvent appearance empirical formula MH+313 yellow oil C25H28ClF3N2O4\1 499 C2HF3O2 314 yellow oil C17H24N2O4321 315 30% EtOAc/hex dark yellow C21H31N3O6 422 gum 316 20-50% paleyellow oil C27H36N2O5 469 EtOAc/hex 317 pale yellow oil C31H44N2O5 525318 gum C24H30N2O4 411 319 pale yellow oil C24H30N2O4 411 320 2% MeOH/yellow oil C25H32N2O5 441 CH2Cl2 321 yellow oil C26H32N2O6\1 469 C2H4O2322 25-50% clear residue C28H32N2O4 461 EtOAc/hex 323 yellow oilC25H30N2O6 455 324 yellow oil C26H32N2O6 469

[0160] TABLE 19A

EPO/EBP-Ig cpd % inh @ 50 μM R¹ R² R³ R⁴ 325  6 Me OPh H CH2CH2CO2H 326 0 H OPh H CH2CH2CO2H 327 11 Me H Ph CH2CH2CO2H 328 33 Me 3,4-Cl2-C6H3OH CH2CH2CO2H 329 13 H H Ph CH2CH2CO2H 330 12 H 3-CF3-C6H4O H CH2CH2CO2H331 18 H 4-t-Bu-C6H4O H CH2CH2CO2H 332 17 H 3,4-Cl2-C6H3O H CH2CH2CO2H333 16 Me 3,4-benzo CH2CH2CO2-t-Bu 334  6 Me OPh H CH2CH2CO2-t-Bu 335 25Me H Ph CH2CH2CO2-t-Bu 336 32 Me 3,4-Cl2-C6H3O H CH2CH2CO2-t-Bu 337  0 HOPh H CH2CH2CO2-t-Bu 338 23 t-Bu 3-CF3-C6H4O H CH2CH2CO2-t-Bu 339 10t-Bu 4-t-Bu-C6H4O H CH2CH2CO2-t-Bu 340 14 H H Ph CH2CH2CO2-t-Bu 341 19 H3,4-Cl2-C6H3O H CH2CH2CO2-t-Bu

[0161] TABLE 19B MS, cpd MPLC solvent appearance empirical formula MH+325 off-white solid C21H23NO5\1 370 C2F3HO2 326 fluffy white C20H21NO5\1356 solid C2HF3O2 327 off-white solid C21H23NO4\1 354 C2F3HO2 328 amberoil C21H21C12NO5\1 438 C2F3HO2 329 amber solid C20H21NO4\1 340 C2HF3O2330 amber oil C21H20F3NO5\1 424 C2HF3O2 331 amber oil C24H29NO5\1 412C2HF3O2 332 amber oil C20H19CL2NO5\1 424 C2HF3O2 333 10-25% EtOAc/hexyellow oil C23H29NO4 384 334 10-30% EtOAc/hex pale yellow oil C25H31NO5426 335 1:5 EtOAc/hex yellow oil C25H31NO4 410 336 1:5 EtOAc/hex yellowoil C25H29C12NO5 494 337 white powder C24H29NO5\0.4 412 H2O 338 1:5EtOAc/hex yellow oil C29H36F3NO5 536 339 1:5 EtOAc/hex yellow oilC32H45NO5 524 340 yellow solid C24H29NO4 396 341 white solidC24H27C12NO5 480

[0162] TABLE 20A

EPO/EBP-Ig cpd % inh @ 50 μM R¹ R² R³ R⁴ 342  0 Me H Ph CH2OH 343 37 Me4-t-Bu-C6H4O H CH2OH 344  4 Me 3-CF3-C6H4O H CH2OH 345 40 Me3,4-Cl2-C6H3O H CH2OH 346 28 Me OPh H CH2OH 347 23 H OPh H CH2OH 348 21H H Ph CH2OH 349 23 H 3,4-Cl2-C6H3O H CH2OH 350 23 H 3-CF3-C6H4O H CH2OH351 29 H 4-t-Bu-C6H4O H CH2OH 352  8 Me OPh H CH2O-t-Bu 353 24 Me H PhCH2O-t-Bu 354 31 Me 3,4-Cl2-C6H3O H CH2O-t-Bu 355 22 Me 3-CF3-C6H4O HCH2O-t-Bu 356 23 Me 4-t-Bu-C6H4O H CH2O-t-Bu 357 12 H 3-CF3-C6H4O HCH2O-t-Bu

[0163] TABLE 20B MS, cpd MPLC solvent appearance empirical formula MH+342 off-white solid C19H21NO3\1 312 C2F3HO2 343 amber oil C23H29NO4\1384 C2F3HO2 344 amber oil C20H20F3NO4\1 396 C2F3HO2 345 amber oilC19H19C12NO4\1 396 C2F3HO2 346 EtOAc pale yellow oil C19H21NO4 328 347amber oil C18H19NO4\1 314 C2HF3O2 348 yellow solid C18H19NO3\1 298C2HF3O2 349 amber oil C18H17C12NO4\1 382 C2HF3O2 350 amber oilC19H18F3NO4\1 382 C2HF3O2 351 amber oil C22H27NO4\1 370 C2HF3O2 35210-30% EtOAc/hex pale yellow oil C23H29NO4 384 353 20% EtOAc/hexoff-white solid C23H29NO3 368 354 20% EtOAc/hex yellow oil C23H27C12NO4452 355 20% EtOAc/hex yellow oil C24H28F3NO4 452 356 20% EtOAc/hexyellow oil C27H37NO4 440 357 white solid C23H26F3NO4 438

[0164] TABLE 21A

EPO/EBP-Ig cpd % inh @ 50 μM R¹ R² R³ R⁴ 358  0 H H CF3 (s)-CH(OH)CH3359 25 Me CO2Me H (s)-CH(OMe)CH3 360 18 Me H H Bn 361 24 Me CO2Me H Bn362  0 H H CF3 CH2(4-HOC6H4) 363 33 Me CO2Me H CH2(4-MeOC6H4) 364 16 MeH H CH2(indol-3-yl) 365  0 H H CF3 CH2CH2SMe 366 38 Me CO2Me H CH2CO2Me367  0 H H CF3 CH2CONH2 368 40 H CO2Me H CH2SBn 369 12 H H CF3 i-Bu 370 0 H H CF3 i-Pr 371 16 Me CO2Me H i-Pr 372  0 Me H H Me

[0165] TABLE 21B MS, cpd MPLC solvent appearance empirical formula MH+358 amber oil C14H16F3NO3\1 304 C2HF3O2 359 amber oil C17H23NO5\1 322C2H4O2 360 20% EtOAc/hex light-yellow oil C19H21NO2 296 361 amber oilC22H25NO5\1 354 C2H4O2 362 amber oil C19H18F3NO3\1 366 C2HF3O2 363 amberoil C22H25NO5\1 384 C2H4O2 364 1:2 EtOAc/hex tan solid C21H22N2O2 335365 amber oil C15H18F3NO2S\1 334 C2HF3O2 366 amber oil C17H21NO6\1 336C2H4O2 367 amber oil C14H15F3N2O3\1 317 C2HF3O2 368 amber oilC22H25NO4S\1 400 C2H4O2 369 amber oil C17H22F3NO2\1 316 C2HF3O2 370amber oil C15H18F3NO2\1 302 C2HF3O2 371 amber oil C17H23NO4\1 306 C2H4O2372 20% EtOAc/hex yellow oil C13H17NO2\0.10 220 C4H8O2

What is claimed is:
 1. A compound of Formula I

I wherein: R¹ is the side chain of a natural or unnatural α-amino acids,where if said side chain contains a protectable group, that group may beprotected with a member of the group consisting of succinyl, glutaryl,3,3-dimethylglutaryl, C₁₋₅alkyl, C₁₋₅alkoxycarbonyl, acetyl,N-(9-fluorenylmethoxycarbonyl), trifluoroacetyl,omega-carboxyC₁₋₅alkylcarbonyl, t-butoxycarbonyl, benzyl,benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, phenylsulfonyl, ureido,t-butyl, cinnamoyl, trityl, 4-methyltrityl,1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl, tosyl,4-methoxy-2,3,6-trimethylbenzenesulfonyl, phenylureido, and substitutedphenylureido (where the phenyl substituents are phenoxy, halo,C₁₋₅alkoxycarbonyl); R² and R³ may be taken together to form asix-membered aromatic ring which is fused to the depicted ring, or areindependently selected from the group consisting of hydrogen, C₁₋₅alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, amino,phenyl, phenoxy, phenylC₁₋₅alkyl, phenyl C₁₋₅alkoxy, substituted phenyl(where the substituents are selected from C₁₋₅alkyl, C₁₋₅ alkoxy,hydroxy, halo, trifluoromethyl, nitro, cyano, and amino), substitutedphenoxy (where the substituents are selected fromC₁₋₅ alkyl, C₁₋₅alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano, and amino),substituted phenylC₁₋₅alkyl (where the substituents are selected fromC₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano,and amino), substituted phenylC₁₋₅alkoxy (where the substituents areselected from C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl,nitro, cyano, and amino), and substituted amino (where the substituentsare selected from one or more members of the group consisting ofC₁₋₅alkyl, halosubstitutedC₁₋₅alkyl, C₁₋₅alknyl, C₁₋₅alkenyl, phenyl,phenylC₁₋₅alkyl, C₁₋₅alkylcarbonyl, halo substituted C₁₋₅alkylcarbonyl,carboxy-C₁₋₅alkyl, C₁₋₅alkoxyC₁₋₅alkyl, cinnamoyl, naphthylcarbonyl,furylcarbonyl, pyridylcarbonyl, C₁₋₅alkylsulfonyl, phenylcarbonyl,phenylC₁₋₅alkylcarbonyl, phenylsulfonyl, phenylC₁₋₅alkylsulfonylsubstituted phenylcarbonyl, substituted phenylC₁₋₅alkylcarbonyl,substituted phenylsulfonyl, substituted phenylC₁₋₅alkylsulfonyl,substituted phenyl, and substituted phenylC₁₋₅alkyl[where the aromaticphenyl, phenylC₁₋₅alkyl, phenylcarbonyl, phenylC₁₋₅alkylcarbonyl,phenylsulfonyl, and phenylC₁₋₅alkylsulfonyl substitutents areindependently selected from one to five members of the group consistingof C₁₋₅alkyl, C₁₋₅alkoxy, hydroxy, halogen, trifluoromethyl, nitro,cyano, and amino]); R⁴ and R⁵ may be taken together to form asix-membered aromatic ring which is fused to the depicted ring, or areindependently selected from the group consisting of hydrogen, C₁₋₅alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, amino,phenyl, phenoxy, phenylC₁₋₅alkyl, phenyl C₁₋₅alkoxy, substituted phenyl(where the substituents are selected from C₁₋₅alkyl, C₁₋₅ alkoxy,hydroxy, halo, trifluoromethyl, nitro, cyano, and amino), substitutedphenoxy (where the substituents are selected from C₁₋₅ alkyl, C₁₋₅alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano, and amino),substituted phenylC₁₋₅alkyl (where the substituents are selected fromC₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano,and amino), substituted phenylC₁₋₅alkoxy (where the substituents areselected from C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl,nitro, cyano, and amino), and substituted amino (where the substituentsare selected from one or more members of the group consisting ofC₁₋₅alkyl, halosubstitutedC₁₋₅alkyl, C₁₋₅alknyl, C₁₋₅alkenyl, phenyl,phenylC₁₋₅alkyl, C₁₋₅alkylcarbonyl, halo substituted C₁₋₅alkylcarbonyl,carboxyC₁₋₅alkyl, C₁₋₅alkoxyC₁₋₅alkyl, cinnamoyl, naphthylcarbonyl,furylcarbonyl, pyridylcarbonyl, C₁₋₅alkylsulfonyl, phenylcarbonyl,phenylC₁₋₅alkylcarbonyl, phenylsulfonyl, phenylC₁₋₅alkylsulfonylsubstituted phenylcarbonyl, substituted phenylC₁₋₅alkylcarbonyl,substituted phenylsulfonyl, substituted phenylC₁₋₅alkylsulfonyl,substituted phenyl, and substituted phenylC₁₋₅alkyl [where the aromaticphenyl, phenylC₁₋₅alkyl, phenylcarbonyl, phenylC₁₋₅alkylcarbonyl,phenylsulfonyl, and phenylC₁₋₅alkylsulfonyl substitutents areindependently selected from one to five members of the group consistingof C₁₋₅alkyl, C₁₋₅alkoxy, hydroxy, halogen, trifluoromethyl, nitro,cyano, and amino]); W is selected from the group consisting of —CH═CH—,—S—, and —CH═N—; Q is selected from the group consisting of —CH═CH—,—S—, and —CH═N—; X is selected from the group consisting of carbonyl,C₁₋₅alkyl, C₁₋₅alkenyl, C₁₋₅alkenylcarbonyl, and (CH₂)_(m)—C(O)— where mis 2-5; Y is selected from the group consisting of carbonyl, C₁₋₅alkyl,C₁₋₅alkenyl, C₁₋₅alkenylcarbonyl, and (CH₂)_(m)—C(O)— where m is 2-5; nis 1, 2, or 3; Z is selected from the group consisting of hydroxy, C₁₋₅alkoxy, phenoxy, phenylC₁₋₅alkoxy, amino, C₁₋₅alkylamino,diC₁₋₅alkylamino, phenylamino, phenylC₁₋₅alkylamino, piperidin-1-ylsubstituted piperidin-1-yl (where the substituents are selected from thegroup consisting of C₁₋₅alkyl, C₁₋₅alkoxy, halo, aminocarbonyl,C₁₋₅alkoxycarbonyl, and oxo; substituted phenylC₁₋₅alkylamino (where thearomatic substitutents are selected from the group consisting ofC₁₋₅alkyl, C₁₋₅alkoxy, phenylC₁₋₅alkenyloxy, hydroxy, halogen,trifluoromethyl, nitro, cyano, and amino), substituted phenoxy (wherethe aromatic substitutents are selected from the group consisting ofC₁₋₅alkyl, C₁₋₅alkoxy, hydroxy, halogen, trifluoromethyl, nitro, cyano,and amino), substituted phenylC₁₋₅alkoxy (where the aromaticsubstitutents are selected from the group consisting of C₁₋₅alkyl,C₁₋₅alkoxy, hydroxy, halogen, trifluoromethyl, nitro, cyano, and amino),—OCH₂CH₂(OCH₂CH₂)_(s)OCH₂CH₂O—, —NHCH₂CH₂(OCH₂CH₂)_(s)OCH₂CH₂NH—,—NH(CH₂)_(p)O(CH₂)_(q)O(CH₂)_(p)NH—, —NH(CH₂)_(q)NCH₃(CH₂)_(s)NH—,—NH(CH₂)_(s)NH—, and (NH(CH₂)_(s))₃N, where s, p, and q areindependently selected from 1-7 with the proviso that if n is 2, Z isnot hydroxy, C₁₋₅ alkoxy, amino, C₁₋₅alkylamino, diC₁₋₅alkylamino,phenylamino, or phenylC₁₋₅alkylamino, piperidin-1-yl with the furtherproviso that if n is 3, Z is (NH(CH₂)_(s))₃N. and salts thereof.
 2. Thecompounds of claim 1 wherein said compound binds to the EPO receptor. 3.A method for modulating EPO receptor, comprising contacting the EPOreceptor with an amount of the compound of claim
 1. 4. A method fortreating a disease or condition mediated by EPO receptor comprisingadministering an effective amount of the compound of claim
 1. 5. Apharmaceutical composition comprising the compound of claim
 1. 6. An EPOreceptor modulating compound of the formula

wherein: R¹ is the side chain of a natural or unnatural α-amino acids,where if said side chain contains a protectable group, that group may beprotected with a member of the group consisting of succinyl, glutaryl,3,3-dimethylglutaryl, C₁₋₅alkyl, C₁₋₅alkoxycarbonyl, acetyl,N-(9-fluorenylmethoxycarbonyl), trifluoroacetyl,omega-carboxyC₁₋₅alkylcarbonyl, t-butoxycarbonyl, benzyl,benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, phenylsulfonyl, ureido,t-butyl, cinnamoyl, trityl, 4-methyltrityl,1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl, tosyl,4-methoxy-2,3,6-trimethylbenzenesulfonyl, phenylureido, and substitutedphenylureido (where the phenyl substituents are phenoxy, halo,C₁₋₅alkoxycarbonyl); R¹ and R³ may be taken together to form asix-membered aromatic ring which is fused to the depicted ring, or areindependently selected from the group consisting of hydrogen, C₁₋₅alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, amino,phenyl, phenoxy, phenylC₁₋₅alkyl, phenyl C₁₋₅alkoxy, substituted phenyl(where the substituents are selected from C₁₋₅alkyl, C₁₋₅ alkoxy,hydroxy, halo, trifluoromethyl, nitro, cyano, and amino), substitutedphenoxy (where the substituents are selected fromC₁₋₅ alkyl, C₁₋₅alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano, and amino),substituted phenylC₁₋₅alkyl (where the substituents are selected fromC₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano,and amino), substituted phenylC₁₋₅alkoxy (where the substituents areselected from C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl,nitro, cyano, and amino), and substituted amino (where the substituentsare selected from one or more members of the group consisting ofC₁₋₅alkyl, halosubstitutedC₁₋₅alkyl, C₁₋₅alknyl, C₁₋₅alkenyl, phenyl,phenylC₁₋₅alkyl, C₁₋₅alkylcarbonyl, halo substituted C₁₋₅alkylcarbonyl,carboxyC₁₋₅alkyl, C₁₋₅alkoxyC₁₋₅alkyl, cinnamoyl, naphthylcarbonyl,furylcarbonyl, pyridylcarbonyl, C₁₋₅alkylsulfonyl, phenylcarbonyl,phenylC₁₋₅alkylcarbonyl, phenylsulfonyl, phenylC₁₋₅alkylsulfonylsubstituted phenylcarbonyl, substituted phenylC₁₋₅alkylcarbonyl,substituted phenylsulfonyl, substituted phenylC₁₋₅alkylsulfonyl,substituted phenyl, and substituted phenylC₁₋₅alkyl[where the aromaticphenyl, phenylC₁₋₅alkyl, phenylcarbonyl, phenylC₁₋₅alkylcarbonyl,phenylsulfonyl, and phenylC₁₋₅alkylsulfonyl substitutents areindependently selected from one to five members of the group consistingof C₁₋₅alkyl, C₁₋₅alkoxy, hydroxy, halogen, trifluoromethyl, nitro,cyano, and amino]); R⁴ and R⁵ may be taken together to form asix-membered aromatic ring which is fused to the depicted ring, or areindependently selected from the group consisting of hydrogen, C₁₋₅alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, amino,phenyl, phenoxy, phenylC₁₋₅alkyl, phenyl C₁₋₅alkoxy, substituted phenyl(where the substituents are selected from C₁₋₅alkyl, C₁₋₅ alkoxy,hydroxy, halo, trifluoromethyl, nitro, cyano, and amino), substitutedphenoxy (where the substituents are selected from C₁₋₅ alkyl, C₁₋₅alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano, and amino),substituted phenylC₁₋₅alkyl (where the substituents are selected fromC₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano,and amino), substituted phenylC₁₋₅alkoxy (where the substituents areselected from C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl,nitro, cyano, and amino), and substituted amino (where the substituentsare selected from one or more members of the group consisting ofC₁₋₅alkyl, halosubstitutedC₁₋₅alkyl, C₁₋₅alknyl, C₁₋₅alkenyl, phenyl,phenylC₁₋₅alkyl, C₁₋₅alkylcarbonyl, halo substituted C₁₋₅alkylcarbonyl,carboxyC₁₋₅alkyl, C₁₋₅alkoxyC₁₋₅alkyl, cinnamoyl, naphthylcarbonyl,furylcarbonyl, pyridylcarbonyl, C₁₋₅alkylsulfonyl, phenylcarbonyl,phenylC₁₋₅alkylcarbonyl, phenylsulfonyl, phenylC₁₋₅alkylsulfonylsubstituted phenylcarbonyl, substituted phenylC₁₋₅alkylcarbonyl,substituted phenylsulfonyl, substituted phenylC₁₋₅alkylsulfonyl,substituted phenyl, and substituted phenylC₁₋₅alkyl [where the aromaticphenyl, phenylC₁₋₅alkyl, phenylcarbonyl, phenylC₁₋₅alkylcarbonyl,phenylsulfonyl, and phenylC₁₋₅alkylsulfonyl substitutents areindependently selected from one to five members of the group consistingof C₁₋₅alkyl, C₁₋₅alkoxy, hydroxy, halogen, trifluoromethyl, nitro,cyano, and amino]); W is selected from the group consisting of —CH═CH—,—S—, and —CH═N—; Q is selected from the group consisting of —CH═CH—,—S—, and —CH═N—; X is selected from the group consisting of carbonyl,C₁₋₅alkyl, C₁₋₅alkenyl, C₁₋₅alkenylcarbonyl, and (CH₂)_(m)—C(O)— where mis 2-5; Y is selected from the group consisting of carbonyl, C₁₋₅alkyl,C₁₋₅alkenyl, C₁₋₅alkenylcarbonyl, and (CH₂)_(m)—C(O)— where m is 2-5; Zis selected from the group consisting of hydroxy, C₁₋₅ alkoxy, phenoxy,phenylC₁₋₅alkoxy, amino, C₁₋₅alkylamino, diC₁₋₅alkylamino, phenylamino,phenylC₁₋₅alkylamino, piperidin-1-yl substituted piperidin-1-yl (wherethe substituents are selected from the group consisting of C₁₋₅alkyl,C₁₋₅alkoxy, halo, aminocarbonyl, C₁₋₅alkoxycarbonyl, and oxo;substituted phenylC₁₋₅alkylamino (where the aromatic substitutents areselected from the group consisting of C₁₋₅alkyl, C₁₋₅alkoxy,phenylC₁₋₅alkenyloxy, hydroxy, halogen, trifluoromethyl, nitro, cyano,and amino), substituted phenoxy (where the aromatic substitutents areselected from the group consisting of C₁₋₅alkyl, C₁₋₅alkoxy, hydroxy,halogen, trifluoromethyl, nitro, cyano, and amino), substitutedphenylC₁₋₅alkoxy (where the aromatic substitutents are selected from thegroup consisting of C₁₋₅alkyl, C₁₋₅alkoxy, hydroxy, halogen,trifluoromethyl, nitro, cyano, and amino),—OCH₂CH₂(OCH₂CH₂)_(s)OCH₂CH₂O—, —NHCH₂CH₂(OCH₂CH₂)_(s)OCH₂CH₂NH—,—NH(CH₂)_(p)O(CH₂)_(q)O(CH₂)_(p)NH—, —NH(CH₂)_(q)NCH₃(CH₂)_(s)NH—,—NH(CH₂)_(s)NH—, and (NH(CH₂)_(s))₃N, where s, p, and q areindependently selected from 1-7 and pharmaceutically acceptable saltsthereof.
 7. An EPO receptor modulating compound of the Formula

wherein: R¹ is the side chain of a natural or unnatural α-amino acids,where if said side chain contains a protectable group, that group may beprotected with a member of the group consisting of succinyl, glutaryl,3,3-dimethylglutaryl, C₁₋₅alkyl, C₁₋₅alkoxycarbonyl, acetyl,N-(9-fluorenylmethoxycarbonyl), trifluoroacetyl,omega-carboxyC₁₋₅alkylcarbonyl, t-butoxycarbonyl, benzyl,benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, phenylsulfonyl, ureido,t-butyl, cinnamoyl, trityl, 4-methyltrityl,1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl, tosyl,4-methoxy-2,3,6-trimethylbenzenesulfonyl, phenylureido, and substitutedphenylureido (where the phenyl substituents are phenoxy, halo, isC₁₋₅alkoxycarbonyl); R² and R³ may be taken together to form asix-membered aromatic ring which is fused to the depicted ring, or areindependently selected from the group consisting of hydrogen, C₁₋₅alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, amino,phenyl, phenoxy, phenylC₁₋₅alkyl, phenyl C₁₋₅alkoxy, substituted phenyl(where the substituents are selected from C₁₋₅alkyl, C₁₋₅ alkoxy,hydroxy, halo, trifluoromethyl, nitro, cyano, and amino), substitutedphenoxy (where the substituents are selected fromC₁₋₅ alkyl, C₁₋₅alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano, and amino),substituted phenylC₁₋₅alkyl (where the substituents are selected fromC₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano,and amino), substituted phenylC₁₋₅alkoxy (where the substituents areselected from C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl,nitro, cyano, and amino), and substituted amino (where the substituentsare selected from one or more members of the group consisting ofC₁₋₅alkyl, halosubstitutedC₁₋₅alkyl, C₁₋₅alknyl, C₁₋₅alkenyl, phenyl,phenylC₁₋₅alkyl, C₁₋₅alkylcarbonyl, halo substituted C₁₋₅alkylcarbonyl,carboxyC₁₋₅alkyl, C₁₋₅alkoxyC₁₋₅alkyl, cinnamoyl, naphthylcarbonyl,furylcarbonyl, pyridylcarbonyl, C₁₋₅alkylsulfonyl, phenylcarbonyl,phenylC₁₋₅alkylcarbonyl, phenylsulfonyl, phenylC₁₋₅alkylsulfonylsubstituted phenylcarbonyl, substituted phenylC₁₋₅alkylcarbonyl,substituted phenylsulfonyl, substituted phenylC₁₋₅alkylsulfonyl,substituted phenyl, and substituted phenylC₁₋₅alkyl[where the aromaticphenyl, phenylC₁₋₅alkyl, phenylcarbonyl, phenylC₁₋₅alkylcarbonyl,phenylsulfonyl, and phenylC₁₋₅alkylsulfonyl substitutents areindependently selected from one to five members of the group consistingof C₁₋₅alkyl, C₁₋₅alkoxy, hydroxy, halogen, trifluoromethyl, nitro,cyano, and amino]); R⁴and R⁵ may be taken together to form asix-membered aromatic ring which is fused to the depicted ring, or areindependently selected from the group consisting of hydrogen, C₁₋₅alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, amino,phenyl, phenoxy, phenylC₁₋₅alkyl, phenyl C₁₋₅alkoxy, substituted phenyl(where the substituents are selected from C₁₋₅alkyl, C₁₋₅ alkoxy,hydroxy, halo, trifluoromethyl, nitro, cyano, and amino), substitutedphenoxy (where the substituents are selected from C₁₋₅ alkyl, C₁₋₅alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano, and amino),substituted phenylC₁₋₅alkyl (where the substituents are selected fromC₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano,and amino), substituted phenylC₁₋₅alkoxy (where the substituents areselected from C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl,nitro, cyano, and amino), and substituted amino (where the substituentsare selected from one or more members of the group consisting ofC₁₋₅alkyl, halosubstitutedC₁₋₅alkyl, C₁₋₅alknyl, C₁₋₅alkenyl, phenyl,phenylC₁₋₅alkyl, C₁₋₅alkylcarbonyl, halo substituted C₁₋₅alkylcarbonyl,carboxyC₁₋₅alkyl, C₁₋₅alkoxyC₁₋₅alkyl, cinnamoyl, naphthylcarbonyl,furylcarbonyl, pyridylcarbonyl, C₁₋₅alkylsulfonyl, phenylcarbonyl,phenylC₁₋₅alkylcarbonyl, phenylsulfonyl, phenylC₁₋₅alkylsulfonylsubstituted phenylcarbonyl, substituted phenylC₁₋₅alkylcarbonyl,substituted phenylsulfonyl, substituted phenylC₁₋₅alkylsulfonyl,substituted phenyl, and substituted phenylC₁₋₅alkyl [where the aromaticphenyl, phenylC₁₋₅alkyl, phenylcarbonyl, phenylC₁₋₅alkylcarbonyl,phenylsulfonyl, and phenylC₁₋₅alkylsulfonyl substitutents areindependently selected from one to five members of the group consistingof C₁₋₅alkyl, C₁₋₅alkoxy, hydroxy, halogen, trifluoromethyl, nitro,cyano, and amino]); W is selected from the group consisting of—CH═CH—,—S—, and —CH═N—; Q is selected from the group consisting of —CH═CH—,—S—, and —CH═N—; X is selected from the group consisting of carbonyl,C₁₋₅alkyl, C₁₋₅alkenyl, C₁₋₅alkenylcarbonyl, and (CH₂)_(m)—C(O)— where mis 2-5; Y is selected from the group consisting of carbonyl, C₁₋₅alkyl,C₁₋₅alkenyl, C₁₋₅alkenylcarbonyl, and (CH₂)_(m)C—(O)— where m is 2-5; Zis selected from the group consisting of phenoxy, phenylC₁₋₅alkoxy,substituted piperidin-1-yl (where the substituents are selected from thegroup consisting of C₁₋₅alkyl, C₁₋₅alkoxy, halo, aminocarbonyl,C₁₋₅alkoxycarbonyl, and oxo; substituted phenylC₁₋₅alkylamino (where thearomatic substitutents are selected from the group consisting ofC₁₋₅alkyl, C₁₋₅alkoxy, phenylC₁₋₅alkenyloxy, hydroxy, halogen,trifluoromethyl, nitro, cyano, and amino), substituted phenoxy (wherethe aromatic substitutents are selected from the group consisting ofC₁₋₅alkyl, C₁₋₅alkoxy, hydroxy, halogen, trifluoromethyl, nitro, cyano,and amino), substituted phenylC₁₋₅alkoxy (where the aromaticsubstitutents are selected from the group consisting of C₁₋₅alkyl,C₁₋₅alkoxy, hydroxy, halogen, trifluoromethyl, nitro, cyano, and amino),—OCH₂CH₂(OCH₂CH₂)_(s)OCH₂CH₂O—, —NHCH₂CH₂(OCH₂CH₂)_(s)OCH₂CH₂NH—,—NH(CH₂)_(p)O(CH₂)_(q)O(CH₂)_(p)NH—, —NH(CH₂)_(q)NCH₃(CH₂)_(s)NH—,—NH(CH₂)_(s)NH—, and (NH(CH₂)_(s))₃N, where s, p, and q areindependently selected from 1-7 and the pharmaceutically acceptablesalts thereof.
 8. An EPO receptor modulating compound of the Formula

wherein: R¹ is the side chain of a natural or unnatural α-amino acids,where if said side chain contains a protectable group, that group may beprotected with a member of the group consisting of succinyl, glutaryl,3,3-dimethylglutaryl, C₁₋₅alkyl, C₁₋₅alkoxycarbonyl, acetyl,N-(9-fluorenylmethoxycarbonyl), trifluoroacetyl,omega-carboxyC₁₋₅alkylcarbonyl, t-butoxycarbonyl, benzyl,benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, phenylsulfonyl, ureido,t-butyl, cinnamoyl, trityl, 4-methyltrityl,1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl, tosyl,4-methoxy-2,3,6-trimethylbenzenesulfonyl, phenylureido, and substitutedphenylureido (where the phenyl substituents are phenoxy, halo,C₁₋₅alkoxycarbonyl); R² and R³ may be taken together to form asix-membered aromatic ring which is fused to the depicted ring, or areindependently selected from the group consisting of hydrogen, C₁₋₅alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, amino,phenyl, phenoxy, phenylC₁₋₅alkyl, phenyl C₁₋₅alkoxy, substituted phenyl(where the substituents are selected from C₁₋₅alkyl, C₁₋₅ alkoxy,hydroxy, halo, trifluoromethyl, nitro, cyano, and amino), substitutedphenoxy (where the substituents are selected fromC₁₋₅ alkyl, C₁₋₅alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano, and amino),substituted phenylC₁₋₅alkyl (where the substituents are selected fromC₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano,and amino), substituted phenylC₁₋₅alkoxy (where the substituents areselected from C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl,nitro, cyano, and amino), and substituted amino (where the substituentsare selected from one or more members of the group consisting ofC₁₋₅alkyl, halosubstitutedC₁₋₅alkyl, C₁₋₅alknyl, C₁₋₅alkenyl, phenyl,phenylC₁₋₅alkyl, C₁₋₅alkylcarbonyl, halo substituted C₁₋₅alkylcarbonyl,carboxyC₁₋₅alkyl, C₁₋₅alkoxyC₁₋₅alkyl, cinnamoyl, naphthylcarbonyl,furylcarbonyl, pyridylcarbonyl, C₁₋₅alkylsulfonyl, phenylcarbonyl,phenylC₁₋₅alkylcarbonyl, phenylsulfonyl, phenylC₁₋₅alkylsulfonylsubstituted phenylcarbonyl, substituted phenylC₁₋₅alkylcarbonyl,substituted phenylsulfonyl, substituted phenylC₁₋₅alkylsulfonyl,substituted phenyl, and substituted phenylC₁₋₅alkyl[where the aromaticphenyl, phenylC₁₋₅alkyl, phenylcarbonyl, phenylC₁₋₅alkylcarbonyl,phenylsulfonyl, and phenylC₁₋₅alkylsulfonyl substitutents areindependently selected from one to five members of the group consistingof C₁₋₅alkyl, C₁₋₅alkoxy, hydroxy, halogen, trifluoromethyl, nitro,cyano, and amino]); R⁴ and R⁵ may be taken together to form asix-membered aromatic ring which is fused to the depicted ring, or areindependently selected from the group consisting of hydrogen, C₁₋₅alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, amino,phenyl, phenoxy, phenylC₁₋₅alkyl, phenyl C₁₋₅alkoxy, substituted phenyl(where the substituents are selected from C₁₋₅alkyl, C₁₋₅ alkoxy,hydroxy, halo, trifluoromethyl, nitro, cyano, and amino), substitutedphenoxy (where the substituents are selected from C₁₋₅ alkyl, C₁₋₅alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano, and amino),substituted phenylC₁₋₅alkyl (where the substituents are selected fromC₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl, nitro, cyano,and amino), substituted phenylC₁₋₅alkoxy (where the substituents areselected from C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl,nitro, cyano, and amino), and substituted amino (where the substituentsare selected from one or more members of the group consisting ofC₁₋₅alkyl, halosubstitutedC₁₋₅alkyl, C₁₋₅alknyl, C₁₋₅alkenyl, phenyl,phenylC₁₋₅alkyl, C₁₋₅alkylcarbonyl, halo substituted C₁₋₅alkylcarbonyl,carboxyC₁₋₅alkyl, C₁₋₅alkoxyC₁₋₅alkyl, cinnamoyl, naphthylcarbonyl,furylcarbonyl, pyridylcarbonyl, C₁₋₅alkylsulfonyl, phenylcarbonyl,phenylC₁₋₅alkylcarbonyl, phenylsulfonyl, phenylC₁₋₅alkylsulfonylsubstituted phenylcarbonyl, substituted phenylC₁₋₅alkylcarbonyl,substituted phenylsulfonyl, substituted phenylC₁₋₅alkylsulfonyl,substituted phenyl, and substituted phenylC₁₋₅alkyl [where the aromaticphenyl, phenylC₁₋₅alkyl, phenylcarbonyl, phenylC₁₋₅alkylcarbonyl,phenylsulfonyl, and phenylC₁₋₅alkylsulfonyl substitutents areindependently selected from one to five members of the group consistingof C₁₋₅alkyl, C₁₋₅alkoxy, hydroxy, halogen, trifluoromethyl, nitro,cyano, and amino]); W is selected from the group consisting of —CH═CH—,—S—, and —CH═N—; Q is selected from the group consisting of —CH═CH—,—S—, and —CH═N—; X is selected from the group consisting of carbonyl,C₁₋₅alkyl, C₁₋₅alkenyl, C₁₋₅alkenylcarbonyl, and (CH₂)_(m)C—(O)— where mis 2-5; Y is selected from the group consisting of carbonyl, C₁₋₅alkyl,C₁₋₅alkenyl, C₁₋₅alkenylcarbonyl, and (CH₂)_(m)—C(O)— where m is 2-5; Zis (NH(CH₂)_(s))₃N and pharmaceutically acceptable salts thereof.
 9. Acompound selected from the group consisting of

and salts thereof.
 10. The compounds of claim 9 wherein said compoundbinds to the EPO receptor.
 11. A method for modulating EPO receptor,comprising contacting the EPO receptor with an EPO receptor modulatingamount of the compound of claim
 9. 12. A method for treating a diseaseor condition mediated by EPO receptor comprising administering aneffective amount of the compound of claim
 9. 13. A pharmaceuticalcomposition comprising the compound of claim 9.